Co-activation of mTOR and STAT3 pathways to promote neuronal survival and regeneration

ABSTRACT

Disclosed herein is a method of promoting sustained survival, sustained regeneration, in a lesioned mature neuron, sustained compensatory outgrowth in a neuron, or combinations thereof. The method comprises contacting the lesioned mature neuron with an effective amount of an inhibitor of PTEN and an effective amount of an inhibitor of SOCS3 to thereby promote survival and/or regeneration and/or compensatory outgrowth of the neuron. Therapeutic methods of treatment of a subject with a neuronal lesion by administration of a therapeutically effective amount of an inhibitor of PTEN and a therapeutically effective amount of an inhibitor of SOCS3, are also disclosed, as are pharmaceutical compositions and devices for use in the methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/342,656, filed on Nov. 3, 2016, now abandoned, which is aContinuation of U.S. application Ser. No. 14/354,694, filed on Apr. 28,2014, issued as U.S. Pat. No. 9,511,036, Dec. 6, 2016, which is a 35U.S.C. 371 National Phase Entry Application of International ApplicationNo. PCT/US12/062973, filed on Nov. 1, 2012, which claims benefit under35 U.S.C. 119(e) of U.S. Provisional Application No. 61/554,277, filedon Nov. 1, 2011, the contents of each of which are incorporated hereinby reference in their entireties.

GOVERNMENTAL SUPPORT

This invention was made with Government support under EY021342 andEY021526 awarded by the National Eye Institute. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of neural regeneration.

BACKGROUND OF THE INVENTION

Axon regeneration failure accounts for permanent functional deficitsfollowing neuronal injury in adult mammals. However, the underlyingmechanisms that control axon regeneration in the adult CNS and PNSremain elusive. A formidable challenge in neural repair in the adultnervous system is the long distances that regenerating axons often needto travel in order to reconnect with their targets. Thus, a sustainedcapacity for axon regeneration is critical for achieving functionalrestoration. Although deletion of either Phosphatase and tensin homolog(PTEN), a negative regulator of mammalian target of rapamycin (mTOR), orsuppressor of cytokine signaling 3 (SOCS3), a negative regulator ofJanus kinase/signal transducers and activators of transcription(JAK/STAT) pathway, in adult retinal ganglion cells (RGCs) individuallypromoted significant optic nerve regeneration, such re-growth taperedoff around two weeks after the crush injury^(1,2). The identification offactors and techniques that promote sustained regeneration to damagedneurons is critical for the development of successful therapeutics.

SUMMARY

One aspect of the invention relates to a method of promoting sustainedsurvival in a lesioned mature neuron, sustained regeneration in alesioned mature neuron, sustained compensatory outgrowth in a matureneuron, or a combination thereof. The method comprises, contacting theneuron with an effective amount of an inhibitor of PTEN and an effectiveamount of an inhibitor of SOCS3 to thereby promote sustained survival,sustained regeneration, and/or sustained compensatory outgrowth of theneuron. In one embodiment, the lesioned mature neuron is the result ofan acute injury. In one embodiment, the acute injury is selected fromthe group consisting of crush, severing, and acute ischemia. In oneembodiment, the lesioned mature neuron is the result of chronicneurodegeneration. In one embodiment of the aforementioned inventions,contacting first occurs within 24 hours of the injury. In one embodimentof the aforementioned inventions, contacting first occurs within 3 daysof the injury. In one embodiment of the aforementioned inventions,contacting first occurs within 6 days of the injury. In one embodimentof the aforementioned inventions, contacting is continued for a periodof time selected from the group consisting of 1 week after initiation, 2weeks after initiation 3 weeks after initiation, 4 weeks afterinitiation, 5 weeks after initiation, 6 weeks after initiation, 7 weeksafter initiation, and 8 weeks after initiation. In one embodiment of theaforementioned inventions, contacting occurs in vivo. In one embodimentof the aforementioned inventions, contacting occurs in vitro. In oneembodiment of the aforementioned inventions, the neuron is human.

Another aspect of the invention relates to a method of treating asubject for a CNS lesion. The method comprises administering to thesubject a therapeutically effective amount of an inhibitor of PTEN and atherapeutically effective amount of an inhibitor of SOCS3, whereinadministering results in contacting one or more target CNS neurons ofthe subject with the inhibitor of PTEN and the inhibitor of SOCS3, tothereby promote sustained survival, sustained regeneration, sustainedcompensatory outgrowth, or a combination thereof in the CNS neurons. Inone embodiment, the subject is a human.

In one embodiment of the aforementioned inventions, the inhibitor ofPTEN 15 selected from the group consisting of (a) potassiumbisperoxo(bipyridine)oxovanadate (V) (bpV(bipy)); (b) dipotassiumbisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (V) (bpV(HOpic)) (c)potassium bisperoxo(1,10-phenanthroline)oxovanadate (V), (bpV(phen));(d) dipotassium bisperoxo(picolinato)oxovanadate (V), (bpV(pic)); and(e) combinations thereof. In one embodiment of the aforementionedinventions, the inhibitor of SOCS3 is selected from the group consistingof SOCS3-specific hpRNA, siRNA, antisense SOCS3, dominant negativeSOCS3, and combinations thereof. In one embodiment of the aforementionedinventions, the CNS lesion results from an acute injury. In oneembodiment, the acute injury is selected from the group consisting ofcrush, severing, and acute ischemia. In one embodiment of theaforementioned inventions, administration first occurs within 24 hoursof the injury. In one embodiment of the aforementioned inventions,administration first occurs within 3 days of the injury. In oneembodiment of the aforementioned inventions, administration first occurswithin 6 days of the injury. In one embodiment, the CNS lesion resultsfrom chronic neurodegeneration. In one embodiment, the CNS lesionresults from a traumatic injury. In one embodiment, the CNS lesionresults from a traumatic brain injury. In one embodiment, the CNS lesionresults from a stroke. In one embodiment, the lesioned CNS neuron is inthe optic nerve. In one embodiment, the CNS lesion results from an acutespinal cord injury. In one embodiment, the lesioned CNS neuron is in thespinal cord of a patient, and the inhibitor is intrathecallyadministered to the patient. In one embodiment, lesioned CNS neuron is asensory neuron.

In one embodiment of the aforementioned inventions, the inhibitor isadministered intravenously. In one embodiment of the aforementionedinventions, the inhibitor is administered intrathecally. In oneembodiment of the aforementioned inventions, the inhibitor isadministered ocularly. In one embodiment of the aforementionedinventions, the inhibitor is administered locally at the neuron. In oneembodiment of the aforementioned inventions, an additional agent isadministered to the subject. In one embodiment the additional agent isselected from the group consisting of inosine, oncomodulin, BNDF, NGF,CNTF, and combinations thereof.

Another aspect of the invention related to a device for promotingsustained survival of a lesioned mature neuron, sustained regenerationof a lesioned mature neuron, compensatory outgrowth of a neuron, or acombination thereof, comprising a reservoir loaded with a premeasuredand contained amount of a therapeutically effective amount of aninhibitor of PTEN and an inhibitor of SOCS3. In one embodiment, thedevice is specifically adapted for implementing the method describedherein.

Another aspect of the invention related to a pharmaceutical compositioncomprising a therapeutically effective amount of an inhibitor of SOCS3and a therapeutically effective amount of an inhibitor of PTEN.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1E show experimental results that indicate the synergisticeffects of double deletion of PTEN and SOCS3 on axon regenerationobserved at 4 weeks after injury. (A) Images of the optic nerve sectionsshowing CTB-labeled axons in AAV-Cre-injected SOCS3^(f/f) with CNTF(SOCS3^(−/−)), PTEN^(f/f)(PTEN^(−/−)), or PTEN^(f/f)/SOCS3^(f/f) withCNTF (PTEN^(−/−)/SOCS3^(−/−)) mice. Asterisks: lesion sites. (B)High-magnification images of the boxed area in (A), which is about1.5-2.0 mm from the lesion sites. (C) Regenerating axons at the opticchiasm. (D) Estimated numbers of regenerating axons. There was asignificant difference between PTEN^(−/−)/SOCS3^(−/−) group and others.♦—PTEN^(−/−)/SOCS3^(−/−); ▪—PTEN^(−/−); ▴—SOCS3^(−/−); X-WT; *: p<0.001,ANOVA, Bonferroni's post hoc test. (E) Percentages of TUJ1-positive RGCsin each group compared to that in the intact retinas. *: p<0.001, ANOVA,Tukey's post hoc test. N=7-8 per group. Error bars, s.d. Scale bars: 200μm.

FIG. 2A-FIG. 2D show experimental results that indicate the synergisticeffects of double deletion of PTEN and SOCS3 on optic nerve regenerationin a delayed treatment paradigm. (A) Scheme of the experiment. (B)Images of the optic nerve sections showing CTB-labeled axons inAAV-Cre-injected SOCS3^(f/f) with CNTF (SOCS3^(−/−)), PTEN (PTEN^(−/−)),or PTEN^(f/f)/SOCS3^(f/f) with CNTF (PTEN^(−/−)/SOCS3^(−/−)) mice.Asterisks: lesion sites. (C) Extensive axon regeneration is only evidentin double mutants. Top panel shows the entire optic nerve up to thechiasm. Bottom panels show high-magnified areas (A and B) as indicatedin the top panel. OX: optic chiasm. (D) Estimated numbers ofregenerating axons. At all distances quantified, there was a significantdifference between the PTEN^(−/−)/SOCS3^(−/−) (represented by the topline on the graph) group and the remaining groups (PTEN^(−/−); SOCS3−/−;WT, respectively represented as the next lower lines on the graph, indescending order). *: p<0.001, ANOVA, Bonferroni's post hoc test. N=5-6per group. Error bars, s.d. Scale bars: 100 μm.

FIG. 3A-FIG. 3D show experimental results that indicate STAT3 in axonregeneration is induced by SOCS3 deletion. (A) Images showing signalsdetected with TUJ1 or anti-p-STAT3 antibodies in the retinal sectionsfrom intact or 1-day-post-injury mice. (B). Percentage of RGCs withnuclear phospho-STAT3 signals. N=3-4 per group. *: p<0.001, ANOVA,Dunnett's post hoc test. (C, D) Images (C) and quantification (D) ofoptic nerve sections showing regenerating axons in each group at 14 dayspost-injury. By Bonferroni's post hoc test, regenerating axons inSOCS3^(−/−)/STAT3^(−/−) double mutants were significantly less thanthose in the SOCS3^(−/−) mutants at 0.2-2.0 mm from lesion site (p<0.01;N=5 per group). ▴—SOCS3^(−/−); ♦—SOCS3^(−/−)/STAT3^(−/−); ▪—STAT3^(−/−);X-WT; Error bars, s.d. Scale bars: 50 μm in (A), 100 μm in (C).

FIG. 4A-FIG. 4D show experimental results that indicate the independenceof PTEN- and SOCS3-regulated pathways. (A, B) Images of optic nervesections from PTEN^(−/−) and various PTEN^(−/−) combined groups (A) orSOCS3^(−/−) mutants with or without rapamycin treatment (B) at 14 dayspost-injury. (C, D) Quantification of regenerating axons shown in (A)and (B) respectively. (C) Axon regeneration in either (▪)PTEN^(−/−)/gp130^(−/−) or (▴) PTEN^(−/−)/STAT3^(−/−) group wascomparable to that in (♦) PTEN^(−/−), but was significantly reduced inthe (+) PTEN^(−/−) group with rapamycin (p<0.01, ANOVA, Bonferroni'spost hoc test; N=5 per group) (●) WT; (X) STAT3^(−/−). (D) Rapamycintreatment did not significantly reduce the number of regenerating axonsin (♦) SOCS3^(−/−) mice (N=6 per group) (▪) SOCS3^(−/−) plus rapamycin;(▴) WT. Error bars, s.d. Scale bars: 100 μm.

FIG. 5 shows experimental results of CST sprouting indicatesco-inhibition of PTEN and SOCS3 produced synergy in promotingcompensatory sprouting from intact and spared axons after partialinjury. Left panel (wild type) shows BDA labeled CST fibers were rare inthe denervated side of spinal cord. Central panel (SOCS3^(−/−)) showsSOCS3 deletion promoted robust CST sprouting into the denervated side ofspinal cord. Right panel SOCS3^(−/−) and PTEN^(−/−)): shows SOCS3 andPTEN double deletion synergistically promoted CST sprouting. C6-8 spinalcord level.

FIG. 6A-FIG. 6C show experimental results that indicate axonregeneration is observed at 2 weeks after an optic nerve injury. (A)Representative confocal images of the optic nerve sections showing thatCTB labeled axons in AAV-Cre-injected SOCS3^(f/f) with CNTF(SOCS34^(−/−)), PTEN^(f/f) (PTEN^(−/−)), or PTEN^(f/f)/SOCS3^(f/f) withCNTF (PTEN^(−/−)/SOCS3^(−/−)) mice. Asterisks indicate lesion sites. (B)Quantification of axon regeneration seen in (A) (♦)PTEN^(−/−)/SOCS3^(−/−); (▪) PTEN^(−/−); (▴) SOCS3^(−/−); (X) WT. Thenumbers of regenerating axons in the PTEN^(−/−)/SOCS3^(−/−) group aresignificantly higher than each individual mutant group at all distancemeasured (up to 3 mm away from the lesion site). *: p<0.001, ANOVA,Bonferroni's post hoc test; N=6 per group. Error bars, s.d. (C)Quantification of RGC survival as measured by TUJ1 staining. All threemutant groups had significantly higher numbers of TUJ1+ RGCs afterinjury compared to the injured wild type group. *: p<0.001, ANOVA,Tukey's post hoc test. Error bars, s.d. Scale bars: 200 urn.

FIG. 7A-FIG. 7B are experimental results that show the regeneratingaxons in the brain areas after optic nerve injury in the adult mice withPTEN and SOCS3 double deletion and CNTF treatment at 4 weekspost-injury. (a) A few CTB-labeled axons could be seen in thesuprachiasmatic nuclei (SCN) area (A), as indicated by Dapi nuclearstaining (a′). (B) Regenerating axons in the optic tract at thisanatomical level. (b) More caudally, regenerating axons could be seen ataround the optic tract brain entry zone (A, A′) and occasionally in areamore medially (B). SCN: superachiasmic nuclei; 3rd V: third ventricle;OX: optic chiasm; EZ: entry-zone. Scale bars: 500 urn in (a, a′ and b).50 urn in (A, A′ and B).

FIG. 8A-FIG. 8C are experimental results that show the characterizationof the delayed treatment experiment. (A) Cre-dependent reporter Tomatoexpression in RGCs after intravitreal AAV-Cre injection to Rosa tdTomatomice. (B) Representative retinal whole-mount images with TUJ1 stainingat 3 weeks post-injury. (C) Quantification of RGC survival as measuredby TUJ1 staining. The number from each injured group was compared withthat of intact wild type mice. Between all animal groups subjected toinjury, there was no statistically significant difference in thepercentage of RGC survival. N=5-6. Error bars, s.d. Scale bars: 20 urn.GCL, ganglion cell layer.

FIG. 9A-FIG. 9B are experimental results that show RGC survival at 2weeks post-injury in SOCS3 and STAT3 mutant groups. (A) Representativeretinal whole-mount images with TUJ1 staining at 2 weeks post-injury inAAV-Cre injected mice of SOCS3^(f/f) (SOCS3^(−/−)), STAT3^(f/f)(STAT3^(−/−)) or SOCS3^(f/f)/STAT3^(f/f) (SOCS3^(−/−)/STAT3^(−/−)).Scale bar: 50 urn. (B) Quantification of RGC survival. The numbers ofTUJ1+ RGCs were significantly higher in SOCS3^(−/−) group thanSTAT3^(−/−) and SOCS3^(−/−)/STAT3^(−/−) groups. There was no significantdifference between STAT3^(−/−) and SOCS3^(−/−)/STAT3^(−/−) groups. *:p<0.001, ANOVA, Tukey's post hoc test. Error bars, s.d. N=5 per group.

FIG. 10A-FIG. 10B are experimental results that indicate the effects ofPTEN deletion on phospho-STAT3levels in RGCs at 2 weeks post-injury. (A)Representative images of retinal sections showing the immunoreactivitywith Phospho-STAT3(originally visualized in red, reproduced here inblack and white) or TUJ1(originally visualized in green, reproduced herein black and white) in AAV-Cre injected mice of wild type (WT),PTEN^(f/f) or PTEN^(f/f)/STAT3^(f/f). Scale bar: 50 urn. (B)Quantification of p-STAT3 immunoreactive RGCs. When compared to thePTEN^(−/−) group, the percentages of TUJ1+RGCs with nuclear p-STAT3signals was similar in the WT group, but was significantly reduced inthe PTEN^(−/−)/STAT3^(−/−) group. *, p<0.01, Student's t test. Errorbars, s.d. N=3 per group.

FIG. 11A-FIG. 11D are experimental results that indicate RGC survival invarious animal groups. (A) Representative images of TUJ1-stained retinalwhole mount at 2 weeks post-injury in AAV-Cre injected mice ofPTEN^(f/f) (PTEN^(−/−)), PTEN^(f/f)/STAT3^(f/f)(PTEN^(−/−)/STAT3^(−/−)), PTEN^(f/f)/gp130^(f/f)(PTEN^(f−/−)/gp130^(−/−)), or PTEN^(f/f) with rapamycin treatment. (B)Quantification of RGC survival as shown in (A). When compared to thePTEN^(−/−) group, only PTEN^(−/−) treated with rapamycin hadsignificantly less survived RGCs. *: p<0.001, ANOVA, Dunnett's post hoctest. Error bars, s.d. N=4 per group. (C) Representative images ofTUJ1-stained retinal whole mount at 2 weeks post-injury in AAV-Creinjected mice of SOCS3^(f/f)(SOCS3^(−/−)) with or without rapamycin. (D)Quantification of RGC survival as shown in (C). There was no significantdifference between these two groups. Student's t test. Error bars, s.d.N=4 per group. Scale bars: 50 um.

FIG. 12A-FIG. 12E are experimental results that show isolation of YFP+RGCs for RNA extraction and microarray analysis. (A) Representativeimages of retinal sections from YFP-17 mice detected for fluoresence orTUJ1+ signal showing that most of RGCs are YFP+. Only few non-RGCs wereweakly labeled with YFP in the inner retinal layer. (B-E) RepresentativeFACS plots illustrating the process of isolating YFP+ retinal cellpopulation. (B) Dissociated retinal cells were gated based on both size(forward scatter, FSC, x axis) and surface characteristics (sidescatter, SSC, y axis) to select the RGC neurons (P1). (C) Subsequently,aggregated cells were excluded based on FSC-H vs. FSC-A ratio, andsingle cells were selected (P2). (D) Retinal cells without YFPexpression were used as control to set up the threshold for YFP positivecells in FL1 (FITC, x-axis) and FL2 (PE, y-axis) channels (P3). (E) YFPexpressing cells within the gate (P3) were collected, which showedtypical distribution of fluorescent signal.

FIG. 13A-FIG. 13C are experimental results that show genes significantlydifferent between the double mutant and both of the single mutants andthe wild type controls. Gene expression levels of the sorted RGCs fromthe wild type, single or double mutants at 3 days post-injury weresubjected to microarray analysis. Using q<0.05 as the cutoff criteria(FDR q value, SAM analysis), three lists of genes were obtained: 1.Genes whose expression levels are significantly different between PSC(PTEN/SOCS3 double mutant with crush) and PC (PTEN single mutant withcrush), defined as [PSC vs. PC]; 2. Genes whose expression levels aresignificantly different between PSC and SC (SOCS3 single with crush),defined as [PSC vs. SC]; and 3. Genes whose expression levels aresignificantly different between PSC and wild type crush control (WTC),defined as [PSC vs. WTC]. (A) Highlighted area in the Venn diagramshowing the genes that appear in ALL three lists, defined as [PSC vs.PC] AND [PSC vs. SC] AND [PSC vs. WTC]. The expression levels ofindividual genes in each mutant were shown in the list (B) and the bargraph (C), expressed as the ratios of their expression over that in wildtype crush control (WTC). Value in bold (B) or asterisk (C) indicates asignificant difference from WTC.

FIG. 14A-FIG. 14B show experimental results that indicate genessignificantly altered in the double mutant but NOT in the single mutantswhen compared to the wild type controls. Using the cut-off criteria offold change>1.6 and FDR<0.05 (SAM), three lists were obtained: (1).Genes whose expression levels are significantly different between PSCand WTC, defined as [PSC vs. WTC]; (2). Genes whose expression levelsare significantly different between PC and WTC, defined as [PSC vs.WTC]; (3). Genes whose expression levels are significantly differentbetween SC and WTC, defined as [SC vs. WTC]. Thereafter, Genes in either[PC vs. WTC], or [SC vs. WTC] were EXLUDED from the list of [PSC vs.WTC], and the remaining genes in [PSC vs. WTC] were defined as [PSC vs.WTC] NOT [PC vs. WTC] NOT [SC vs. WTC]. (A). The expression levels ofindividual genes in each mutant, are shown as the ratios of theirexpression over that in wild type crush control (WTC). Value in boldindicates a significant difference from WTC (FDR<0.05, SAM). Someregeneration-associated genes are highlighted and are also listed inFIG. 16. (B). For this gene set, functional annotation clusteringanalysis was performed using DAVID. The functional annotation groupswith similar EASE score, the Fish Exact Probability Value, wereclustered and grouped under the same overall enrichment score. The fivetop-scored clusters with their counts of genes and their percentages tocorresponding categories in the database are given.

FIG. 15A-FIG. 15D show experimental results that indicate genessignificantly different between the double mutant and one of the singlemutants and the wild type control. (A, B) Genes whose expression levelsare significantly different between PSC and PC, PSC and WTC, but NOTbetween PSC and SC, are indicated by the highlighted area in the Venndiagram (A), and defined as [PSC vs. PC] AND [PSC vs. WTC] NOT [PSC vs.SC]. (B) Gene expression levels in each mutant are shown as the ratiosof their expression over that in wild type crush control (WTC). Value inbold indicates a significant difference from WTC (FDR<0.05, SAM). Listedare genes with fold change>1.6 in both [PSC vs. PC] and [PSC vs. WTC].(C, D) Genes whose expression levels are significantly different betweenPSC and SC, PSC and WTC, but NOT between PSC and PC, are indicated bythe highlighted area in the Venn diagram (C), and defined as [PSC vs.SC] AND [PSC vs. WTC] NOT [PSC vs. PC]. (D) Gene expression levels ineach mutant are shown as the ratios of their expression over that inwild type crush control (WTC). Value in bold indicates significantdifference than WTC (FDR<0.05, SAM). Listed are genes with foldchange>1.6 in both [PSC vs. SC] and [PSC vs. WTC]. For both (B) and (D),some regeneration-associated genes were highlighted and are also listedin FIG. 16.

FIG. 16 shows experimental results that indicate the expression levelsof some known regeneration-related genes in PTEN and/or SOCS3 mutants.The expression levels of individual genes in each mutant are shown asthe ratios of their expression over that in wild type crush controls(WTC). In addition to those genes shown in the gene lists describedabove, many genes implicated in PNS axon regeneration, such as Jun,GAP43, Id2, SOX11, ATF3 and galanin, are most significantly altered inthe groups with SOCS3 deletion (SC and PSC groups), consistent with thenotion that JAK/STAT pathway is a critical pathway for PNS axonregeneration. Kruppel-like factors KLF4 and KLF6 showed expressionchanges in opposite directions (although the changes of KLF4 did notreach the level of statistical significance), consistent with proposedfunctions of these regeneration regulators.

FIG. 17A-FIG. 17F shows the presence of mRNAs of different genes inretinal sections detected by in situ hybridization. (A-F) Representativeimages showing the mRNA signals detected by anti-sense probes of Elav14(HuD) (A), KLF6 (B), ZFP40 (C), Dyncl 112 (D), Kif21 a (E), and Kifap3(F) at the retinal sections from intact or 3 days post-injury of wildtype (WT), PTEN^(−/−), SOCS3^(−/−) with CNTF, and PTEN^(−/−)/SOCS3^(−/−)with CNTF mice. N=5-6 per group. Scale bars: 50 um.

FIG. 18A-FIG. 18F show experimental results that indicate SOCS3 deletionin neonatal cortical neurons increases CST sprouting after unilateralpyramidotomy. (A-C) Representative images of cervical 7 (C7) spinal cordtransverse sections from Socs3^(f/f) mice with cortical AAV-Creinjection and a sham injury (A) or with cortical AAV-GFP injection and aleft pyramidotomy (Py) (B) or cortical AAV-Cre injection and a leftpyramidotomy (C). As illustrated in FIG. 22, AAVs were injected into theright sensorimotor cortex of P1 Socs3^(f/f) mice, which then received aleft pyramidotomy or sham lesion at 8 weeks. BDA was injected into theright sensorimotor cortex at 4 weeks post-injury and the mice wereterminated 2 weeks later. (D) Quantification of sprouting axon densityindex (contralateral/ipsilateral). *P<0.01, ANOVA followed byBonferroni's post hoc test. (E) Scheme of quantifying crossing axons atdifferent regions of the spinal cord (Mid: midline, Z1 or Z2: differentlateral positions). (F) Quantification of crossing axons counted indifferent regions of spinal cord normalized against the numbers oflabeled CST axons. *P<0.01, ANOVA followed by Bonferroni's post hoctest. Five mice used in each group. Three sections at the C7 level werequantified per mouse. Scale bar: 500 μm.

FIG. 19A-FIG. 19D show experimental results that indicate SOCS3 deletionin juvenile cortical neurons enhances CST sprouting after leftpyramidotomy. (A-B) Representative images of cervical 7 (C7) spinal cordtransverse sections from CamkII-cre mice crossed with wild type (A) orSocs3^(f/f) (B) with a left pyramidotomy (Py) at the age of 8 weeks. BDAwas injected into the right sensorimotor cortex at 4 weeks post-injuryand the mice were terminated 2 weeks later. (C) Quantification ofsprouting axon density index (contralateral/ipsilateral). *P<0.01,T-test. (D) Quantification of crossing axons counted in differentregions of spinal cord normalized against the numbers of labeled CSTaxons. *P<0.01, T-test. Five mice in each group. Three sections at theC7 level were quantified per mouse. Scale bar: 500 um.

FIG. 20A-FIG. 20G show experimental results that indicate CNTFexpression in the neurons of the spinal cord deprived of CST inputs.(A-F) Representative images of the C7 transverse sections from the adultmice with a sham injury (A, C, and E) or 3 days after a leftpyramidotomy (B, D, and F) stained with anti-CNTF (A, B), anti-NeuN (C,D) antibodies. Merged images are shown in E and F. (G) Highmagnification images from the area boxed in B showing the co-staining ofanti-NeuN and anti-CNTF. Scale bars: 500 μm for A-D and 20 μm for G.

FIG. 21A-FIG. 21E show experimental results that indicate significantenhanced CST sprouting induced by co-deletion of SOCS3 and PTEN. (A-C)Representative images of cervical 7 (C7) spinal cord transverse sectionsfrom Socs3^(f/f)/Pten^(f/f) mice with cortical AAV-Cre injection and asham injury (A) or with cortical AAV-GFP injection and a leftpyramidotomy (Py) (B) or cortical AAV-Cre injection and a leftpyramidotomy (C). The experimental procedures were described in FIG.22G. (D) Quantification of sprouting axon density index(contralateral/ipsilateral). *P<0.01, ANOVA followed by Bonferroni'spost hoc test. (E) Quantifications of crossing axons counted indifferent regions of spinal cord normalized against the numbers oflabeled CST axons. *P<0.01, ANOVA followed by Bonferroni's post hoctest. Five mice used in each group. Three sections at the C7 level werequantified per mouse. Scale bar: 500 μm.

FIG. 22A-FIG. 22G show experimental results that indicate SOCS3 deletionin neonatal cortical neurons increases CST sprouting at thoracic andlumbar levels after unilateral pyramidotomy. (A-D) Representative imagesof T4 (A, C) or L1 (B, D) spinal cord transverse sections fromSocs3^(f/f) mice with cortical AAV-GFP injection (A, B) or AAV-Cre (C,D) and a left pyramidotomy (Py). (E) Quantification of sprouting axondensity index (contralateral/ipsilateral). *P<0.01, T-test. (F)Quantification of crossing axons counted in different regions of spinalcord normalized against the numbers of labeled CST axons. *P<0.01,T-test. Five mice used in each group. Three sections at the C7 levelwere quantified per mouse. Scale bar: 500 μm. (G) Scheme of theexperiments. AAVs were injected into the right sensorimotor cortex of P1Socs3^(f/f) mice, which then received a left pyramidotomy or sham lesionat 8 weeks. BDA was injected into the right sensorimotor cortex at 4weeks post-injury and the mice were terminated 2 weeks later.

FIG. 23A-FIG. 23F show experimental results that indicatecharacterization of the CamkII-Cre line. (A-D) Representative transversespinal cord sections from the CamkII-Cre mice crossed with a foxedTomato reporter at the age of 1 week (A), 2 weeks (B), 3 weeks (C), or 2months (D). In the dorsal column, the Tomato signal is seen at the ageof 2 months, but not 1-3 weeks, consistent with its reported expressionpatterns (Yu et al., 2001). Cre is expressed in the superficial levelsof dorsal spinal cord starting from 3 weeks. Scale bar: 500 μm. (E, F)co-localization of some Cre-expressing cells with NeuN (E) or GFAP (F).Scale bar: 20 μm.

FIG. 24A-FIG. 24H show experimental results that indicateco-localization of CNTF with NeuN+, but not CD68+ or GFAP+ cells. (A-D)Representative images of C7 transverse sections from the adult mice witha sham injury (A, B) or 3 days after a left pyramidotomy (C, D) stainedwith anti-CD68 (A, C) or anti-GFAP (B, D). Scale bar: 500 μm. (E-F) Highmagnification images from the spinal cord of 3 days post-injury showingthe co-staining of anti-CNTF with anti-CD68 (E) or anti-GFAP (F). Scalebar: 20 μm. (G, H) Representative images of the cortical sections fromthe adult mice with a sham injury (G) or 3 days after a leftpyramidotomy (H) stained with anti-CNTF. Scale bar: 100 μm.

FIG. 25A-FIG. 25F show experimental results that indicate CNTF injectedto the spinal cord leading to CST sprouting. (A, B) Representativeimages from the C1 transverse spinal cord sections from the mice SOCS3f/f with cortical AAV-Cre injection at P1 and intraspinal injection ofsaline (A) or CNTF (B) at 8 weeks. Arrows indicates the injection needletrajectory. (C, D) High magnification images from (A, B) highlights themidline areas without (C) or with (D) numerous crossing CST axons. (E,F) No CST sprouts seen at C7 levels of the mice with saline (E) or CNTF(F) injection. Scale bar: 500 μm for A, B, E, and F; 100 μm for C, andD.

FIG. 26A-FIG. 26E show experimental results that indicate furtherenhanced CST sprouting in the lower spinal cord of the mice withco-deletion of PTEN and SOCS3 in cortical neurons. (A-D) Representativeimages of T4 (A, C) or L1 (B, D) spinal cord transverse sections fromPTEN^(f/f)/Socs3^(f/f) mice with cortical AAV-GFP injection (A, B) orAAV-Cre (C, D) and a left pyramidotomy (Py). (E) Quantification ofcrossing axons counted in different regions of spinal cord normalizedagainst the numbers of labeled CST axons, *P<0.01, T-test, Five miceused in each group. Three sections at the C7 level were quantified permouse. Scale bar: 500 μm.

DETAILED DESCRIPTION OF THE INVENTION

Previous work has indicated that inhibition of either PTEN or SOCS3results in limited neuronal regeneration of injured neurons. Remarkably,the experiments disclosed herein indicate that simultaneous inhibitionof both PTEN and SOCS3 enables robust and sustained axon regeneration.PTEN and SOCS3 are also shown to regulate two independent pathways thatact synergistically to promote enhanced axon regeneration. Geneexpression analyses suggest that co-inhibition of PTEN and SOCS3 notonly results in the induction of many growth-related genes, but alsoallows neurons to maintain the expression of a repertoire of genes atthe physiological level after injury. These results indicate thatconcurrent activation of mTOR and STAT3 pathways can sustainlong-distance axon regeneration in adult, a crucial step towardfunctional recovery.

Aspect of the invention relate to the combined inhibition of PTEN andSOCS3 in an injured neuron to induce extended or sustained survival andregeneration following an injury. As such, one aspect of the inventionrelates to a method of promoting sustained survival, sustainedregeneration, or a combination of both, in a lesioned mature neuron. Themethod comprises contacting the lesioned mature neuron with an effectiveamount of an inhibitor of PTEN and an effective amount of an inhibitorof SOCS3 to thereby promote sustained survival, sustained regeneration,or a combination of both, of the neuron.

It has also been observed that combined inhibition of PTEN and SOCS3 inan uninjured target neuron will promote axonal outgrowth of uninjuredneurons to an area of injury and such outgrowth can have a compensatoryrole in recovery of the organism from the injury. Another aspect of theinvention relates to a method of promoting sustained compensatoryoutgrowth of an uninjured target neuron to a region of neuronal lesion,comprising contacting the target neuron with an effective amount of aninhibitor of PTEN and an effective amount of an inhibitor of SOCS3, tothereby promote compensatory outgrowth (e.g. axonal) of the targetneuron to the region of neuronal lesion. In one embodiment, the targetneuron is further contacted with a denervation-induced cytokine (e.g.,CNTF) at a location proximal to the site of neuronal lesion.

Another aspect of the invention relates to a method of treating asubject for a nervous system lesion. The method comprises administeringto the subject a therapeutically effective amount of an inhibitor ofPTEN and a therapeutically effective amount of an inhibitor of SOCS3,wherein administering results in contacting one or more lesioned neuronsand/or the lesion site of the subject with the inhibitor of PTEN and theinhibition of SOCS3, to thereby promote sustained survival, sustainedregeneration, or a combination of both in the injured neurons. Thecontacting occurs at the same time so as to coordinately inhibit bothPTEN and SOCS3.

An effective amount of the inhibitors are contacted with the neuronusing a suitable method sufficient to promote sustained survival of theneuron and/or regeneration and/or sustained compensatory outgrowth ofthe neuronal axon. An effective amount is the amount required to producestatistically significant and reproducible sustained survival, sustainedregeneration, or a combination of both, as compared to an appropriatecontrol. For in vitro methods, the inhibitors are, for example, added tothe culture medium, usually at nanomolar or micromolar concentrations.The respective inhibitors can be added in the same formulation, or indifferent formulations.

For in vivo applications, the inhibitors can be administered to thesubject by any method that results in contacting both a therapeuticallyeffective amount of each to the neuron at relatively the same time,e.g., orally, by intravenous (i.v.) bolus, by i.v. infusion,subcutaneously, intramuscularly, ocularly (intraocularly, periocularly,retrobulbarly, intravitreally, subconjunctivally, topically, by subtenonadministration, etc.), intracranially, intraperitoneally,intraventricularly, intrathecally, by epidural, etc. The respectiveinhibitors can be administered at the same time or at different times,depending upon various factors associated with each inhibitor (e.g.,half life, administration route, etc.). The respective inhibitors can beadministered by the same route of administration or through differentroutes of administration. The administration of the respectiveinhibitors can be for differing prolonged periods, as long as thecombined administration conforms to the time periods specified hereinfor both inhibitors such that their activities on the contacted neuronscompletely or substantially overlap. The respective inhibitors can beadministered in a formulation which contains both inhibitors (apharmaceutical composition, as described herein), or they can be inseparate formulations (separate pharmaceutical compositions) forseparate administration.

Sustained survival of a neuron is indicated by the number of neuronssurviving from a specific injury or condition, as compared to the numberof neurons surviving as a result of the effects of the individualinhibitor (either PTEN or SOCS3), and also by the length of time thesurvival persists, as compared to the length of time survival persistsas a result of the effects of the individual inhibitor (either PTEN orSOCS3). Survival is considered to be sustained if it persists for anextended period of time post-injury (e.g., greater than 2 weekspost-injury, greater than 3 weeks, and greater than 4 weekspost-injury). In one embodiment, greater than 10% of neurons (e.g., 15%,20%, 25%, 30%, 35%, 0%, 45%, 50%, 55%, 60%, 65%, 70% and 75%), survivefor an extended period of time post-injury. In one embodiment, greaterthan 20% of neurons survive for an extended period of time post-injury.

Sustained regeneration or outgrowth is indicated by the number ofneurons (injured and also uninjured) and by extended length of theaxonal outgrowth of the neurons, as compared to the number of neuronsand extended length of the axonal outgrowth of the neurons that resultsfrom the effects of the individual inhibitor (either PTEN or SOCS3), andby the time frame post-injury that the outgrowth occurs, as compared tothe time frame post-injury that outgrowth occurs resulting from theeffects of the individual inhibitor (either PTEN or SOCS3). Sustainedregeneration and axonal outgrowth occurs if greater than 10% or greaterthan 20% (e.g., 15%, 20%, 25%, 30%, 35%, 0%, 45%, 50%, 55%, 60%, 65%,70% and 75%) of the neurons regenerate injured axons or generate newaxons, that extend at least 0.5 mm distal to the lesion epicenter. Inone embodiment, greater than 10% or greater than 20% (e.g., 15%, 20%,25%, 30%, 35%, 0%, 45%, 50%, 55%, 60%, 65%, 70% and 75%) of neuronsregenerate injured axons or generate axons over 1 mm distal to thelesion site. In one embodiment, greater than 10% (e.g., 15%, 20%, 25%,30%, 35%, 0%, 45%, 50%, 55%, 60%, 65%, 70% and 75%) or greater than 20%of neurons regenerate or generate new axons that extend at least 2 mmdistal from the lesion site.

Sustained regeneration and axonal outgrowth is also indicated by asignificant amount of outgrowth occurs on or after 2 weeks post-injury.For example significant outgrowth occurs for up to 3 weeks or 4 weekspost-injury.

Neurons

The methods and compositions described herein are suited for thepromotion of sustained survival, sustained neuronal regeneration andsustained axonal outgrowth of CNS (central nervous system) and PNS(peripheral nervous system) neurons. In one embodiment the neuron is aterminally differentiated neuron. In one embodiment, the neuron is anadult neuron (e.g, in a subject that has reached maturity, such as inhumans older than 18 years). In one embodiment, the neuron isnon-embryonic. In one embodiment, the neuron is in an immature organism(e.g., embryo, infant, child).

All CNS and PNS neurons are suitable for such methods described herein.CNS neurons include, without limitation, a cerebellar granule neuron, oran ocular neuron. In one embodiment, the neuron is the optic nerve. Inone embodiment, the neuron is a sensory neuron (e.g., dorsal rootganglion (DRG) sensory neuron). In one embodiment, the CNS neuron isknown or determined to be under specific PTEN and/or SOCS3 regenerationinhibition. Such determination can be determined by the skilledpractitioner.

As used herein, the term “PNS neurons” is intended to include theneurons commonly understood as categorized in the peripheral nervoussystem, including sensory neurons and motor neurons. The presentinvention provides methods and compositions for preventing and/ortreating peripheral nerve damage (peripheral neuropathy) in a subject.Peripheral nerves such as dorsal root ganglia, otherwise known as spinalganglia, are known to extend down the spinal column. These nerves can beinjured as a result of spinal injury. Such peripheral nerve damageassociated with spinal cord injury can also benefit from neuron axonaloutgrowth produced by the methods described herein.

All mammals are suitable subjects for performance of the methodsdescribed herein. In one embodiment, the mammal is a human, non-humanprimate, companion animal (e.g., dog, cat), livestock animal (e.g.,horse, cow, pig, sheep), or rodent (mouse, rat, rabbit). In oneembodiment, the subject is a non-human primate animal in a model forneurodegeneration or nervous system (CNS or PNS) injury. Neurons derivedfrom said subjects are also suitable for performance of the methodsdescribed herein.

Neuronal Lesions

As used in the art, the term lesion refers to damage (e.g., to a systemor a cell). Damage to a system is evidenced by aberrant function,reduction of function, loss of function of the system, or loss ofessential components (e.g., specialized cells such as neurons). Damageto a specific neuron is also evidenced by aberrant function, loss offunction, reduced function, and/or cell death. Some forms of damage to aneuron can be directly detected (e.g., by visualization as with asevered or crushed neuronal axon). Neuronal lesions can result from avariety of insults, including, injury, toxic effects, atrophy (e.g., dueto lack of trophic factors). Injuries that typically cause neuronallesions include, without limitation, severing and crushing.

A neuronal lesion, as the term is used herein, results from damage tothe neuron. Such damage may be complete loss of a neuron, or loss of apart of the neuron (e.g., an axon). Such damage may results from acuteor traumatic injury to the neuron (e.g., crush, severing) such as theresult of external trauma to the subject (e.g., contusion, laceration,acute spinal cord injury, traumatic brain injury, cortical impact,etc.). Acute or traumatic injury to a neuron can also result from anacute condition, such as stroke, that results in acute ischemia to theneuron resulting in acute damage. The specific location of neuronaldamage will vary with the specific cause of the damage, and the specificindividual. In one embodiment of the invention described herein, thelesioned CNS neuron is located in CNS white matter, particularly whitematter that has been subjected to traumatic injury. The specificlocation of a lesion to a specific neuron will vary with respect to theinjury. In one embodiment, the lesion is in the axon or dendrite of aneuron.

Damage to a neuron may also be incurred from a chronic injury (e.g.,repetitive stress injury) or condition (e.g., chronic inflammation ordisease). Chronic injury leads to neurodegeneration such as caused byneurotoxicity or a neurological disease or disorder (e.g. Huntington'sdisease, Parkinson's disease, Alzheimer's disease, multiple systematrophy (MSA), etc.).

In one embodiment of the invention, damage results from an ocular injuryor disorder (e.g. toxic amblyopia, optic atrophy, higher visual pathwaylesions, disorders of ocular motility, third cranial nerve palsies,fourth cranial nerve palsies, sixth cranial nerve palsies, internuclearophthalmoplegia, gaze palsies, eye damage from free radicals, etc.), oran optic neuropathy (e.g. ischemic optic neuropathies, toxic opticneuropathies, ocular ischemic syndrome, optic nerve inflammation,infection of the optic nerve, optic neuritis, optic neuropathy,papilledema, papillitis, retrobulbar neuritis, commotio retinae,glaucoma, macular degeneration, retinitis pigmentosa, retinaldetachment, retinal tears or holes, diabetic retinopathy, iatrogenicretinopathy, optic nerve drusen, etc.).

Damage to a neuron can be detected by the skilled practitioner through avariety of assays known in the art. Loss of function assays can be usedto determine neuronal damage. Physical damage to the neuron (e.g.,axonal crushing or severing) can sometimes be observed diagnosticallythrough routine methods. One way to detect a lesion is through detectionof axotomy-induced stress and/or pathology-induced down-regulation ofprotein translation (e.g., detected directly, indirectly, or inferred).

Diseases and Disorders

The methods and compositions of the invention are useful for treatmentof diseases or disorders resulting from or leading to the neuronallesions described herein. For example, the methods and compositionsdescribed herein can be used specifically to treat damage associatedwith peripheral neuropathies including, but not limited to, thefollowing: diabetic neuropathies, virus-associated neuropathies,including acquired immunodeficiency syndrome (AIDS) related neuropathy,infectious mononucleosis with polyneuritis, viral hepatitis withpolyneuritis; Guillian-Barre syndrome; botulism-related neuropathy;toxic polyneuropathies including lead and alcohol-related neuropathies;nutritional neuropathies including subacute combined degeneration;angiopathic neuropathies including neuropathies associated with systemiclupus erythematosis; sarcoid-associated neuropathy; carcinomatousneuropathy; compression neuropathy (e.g. carpal tunnel syndrome) andhereditary neuropathies, such as Charcot-Marie-Tooth disease, peripheralnerve damage associated with spinal cord injury can also be treated withthe present method. The subject is treated in accordance with thepresent method for peripheral nerve damage as the result of peripheralneuropathies, including those listed above. Subjects at risk fordeveloping such peripheral nerve damage are also so treated.

PTEN Inhibitors

A variety of PTEN inhibitors suitable for use in the methods andcompositions described herein are known in the art. Some suitable PTENinhibitors, such as vanadium-based PTEN inhibitors or siRNA, aredescribed in U.S. Published Patent Application No. 2009/0305333. Oneexample of a PTEN inhibitor is SF1670 (Cellagen Technology C7316).Another example of a PTEN inhibitor is 4-hydroxynonenal. Yet anotherexample of a PTEN inhibitor is P-REX2a. In one embodiment, the PTENinhibitor is a vanadium-based PTEN inhibitor, such as sold commerciallyby Calbiochem, EMD/Merck, including (a) potassiumbisperoxo(bipyridine)oxovanadate (V) (bpV(bipy)); (b) dipotassiumbisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (V) (bpV(HOpic)); (c)potassium bisperoxo(1,10-phenanthroline)oxovanadate (V), (bpV(phen));and (d) dipotassium bisperoxo(picolinato)oxovanadate (V), (bpV(pic)).Alternative, suitable PTEN inhibitors include PTEN inhibitor compoundsof formulas I-XIV as described in WO2005/097119; vanadium-based PTENinhibitors described in US20070292532 and by Rosivatz et al. 2006 (ACSChem. Biol. 1(12) 780-790); the 1,4-naphthoquinone derivative, shikonin,described by Nigorikawa et al. (Mol Pharmacol 70:1143-1149, 2006); andmenadione (vitamin K3) as described by Yoshikawa et al., Biochim BiophysActa. 2007 April; 1770(4):687-93. PTEN inhibition assays for generalscreening (to identify and confirm alternative, suitable inhibitors) andIC50 determinations are known in the art, e.g. WO 2005/097119. SuitablePTEN inhibitors are also described in WO 2007/0203098, including allrecited genera, subgenera and species disclosed and as described thereinincluding, without limitation, I) Ascorbic acid-based PTEN inhibitors,II) 1,2,3-triazole PTEN inhibitors (such as described in WO02/32896),III) Diamide PTEN inhibitors, IV) Aryl imidazole Carbonyl PTENinhibitors, V) Polyamide PTEN inhibitors, VII)1,10-phenanthroline-5,6-dione PTEN inhibitors, VIII) substitutedphenathrene-9-10-dione PTEN inhibitors, IX) Isatin PTEN inhibitors, X)substituted phenanthren-9-ol PTEN inhibitors, XI) substitutednaphthalene-1,2-dione PTEN inhibitors, XII) substitutednaphthalene-1,4-dione PTEN inhibitors, XIII) Vanadate-Based PTENInhibitors 1. Potassium Bisperoxo(bipyridine) oxovanadate (V) 2.Dipotassium Bisperoxo (5-hydroxypyridine-2carboxyl) oxovanadate (V) 3.Dipotassium Bisperoxo (picolinato)oxovanadate (V) 4.Monoperoxo(picolinato) oxovanadate(V) 5. Potassiun Bisperoxo(1,10-phenanthroline) oxovanadate (V) 6. bis(N,N-Dimethylhydroxamido)hydroxooxovanadate, XIV) T1-loop binding element containing PTENinhibitors. The PTEN inhibitors may contain a group that exists atphysiological pH in significantly anionic form, such as at least 5% ofthe molecular species at pH of 7.4 are anionic charged. Such anionicgroups can bind to PTEN in the T1 loop of the peptide structure insolution.

PTEN-specific antibody and intrabody inhibitors may also be used, suchas have been intrabodies for the therapeutic suppression of a variety ofneurodegenerative pathologies, e.g. Messer et al. Expert Opin Biol Ther.2009 September; 9(9):1189-97.

In one embodiment the PTEN inhibitor is specifically designed and/ortargetted to facilitate delivery to the interior of the targetneuron(s).

PTEN can be effectively inhibited by targeting one or more components ofthe PTEN cell signalling pathway. Examples of such components include,without limitation, phosphatase and tensin homologue (PTEN), glycogensynthase kinase 3 beta (GSK3β), and AKT (also referred to as proteinkinase B (PKB)), such as with compounds that activate aphosphoinositide-3 kinase (PI3K) pathway, for example, inhibitors ofPTEN, inhibitors of GSK3β, or activators of AKT (e.g., as described inU.S. Patent Application Publication 2011/0189308). The use of variouscombinations of PTEN inhibitors or combinations of inhibition approachesis also envisioned.

SOCS3 Inhibitors

Various inhibitors of suppressor of cytokine signaling 3 (SOCS3) areknown in the art. The inhibitor may specifically bind or compete withthe SOCS-3 gene, transcript or translate (protein). Suitable inhibitorsinclude, without limitation, SOCS3-specific polynucleotides and PNAstargeting the SOCS3 gene or transcripts, and include SOCS3-specifichpRNA, siRNA, and antisense polynucleotides. Materials and methods formaking and using such polynucleotides are known in the art, includingdesign and cloning strategies for constructing suitable SOCS3 shRNAexpression vectors (e.g. McIntyre et al., BMC Biotechnol. 2006; 6:1),and suitable antisense SOCS3 cDNAs (Owaki, et al., J. Immunol. 2006 Mar.1; 176(5):2773-80). Suitable SOCS3-specific polynucleotides targetingthe SOCS3 gene or transcripts are also commercially available fromseveral vendors including OriGene (Rockville Md.) such as vectorpRFP-C-RS and pGFP-V-RS, human 29mer shRNA constructs against SOCS3 inpRFP-C-RS and pGFP-V-RS vectors, respectively. SOCS3 specific siRNA isalso widely commercially available, e.g. Santa Cruz Biotechnology, Inc.Examples of specific SOCS3 specific siRNA to inhibiti SOCS3 are providedin U.S. Patent Application Publication 2011/0124706.

Suitable inhibitors also include SOCS3-based polypeptides like dominantnegative SOCS3 peptides and proteins, such as SOCS3 (F25A) (e.g. Owaki,et al., J. Immunol. 2006 Mar. 1; 176(5):2773-80), which contains a pointmutation in the kinase inhibitory region of SOCS3.

SOCS3-specific antibody and intrabody inhibitors may also be used, suchas have been intrabodies for the therapeutic suppression of a variety ofneurodegenerative pathologies, e.g. Messer et al. Expert Opin Biol Ther.2009 September; 9(9):1189-97. A SOCS3 antibody is commercially available(MyBioSource, Catalog # MBS242513).

The structural determination of SOSC3 has also facilitated developmentof small-molecule SOCS3 specific inhibitors, e.g. Babon et al., J MolBiol. 2009 Mar. 20; 387(1):162-74; Babon et al., Mol Cell 2006 Apr. 21;22 (2) 205-16. Structure-based SAR yield chemically diverse smallmolecule SOCS3 inhibitors at micro- and nanomolar activity.

In one embodiment the SOCS3 inhibitor is specifically designed and/ortargeted to facilitate delivery the target neuron(s) cell interior.

SOCS3 inhibition is readily assayed by specific techniques, such asimmunocytochemistry. Because SOCS3 up-regulation occurs after CNTFtreatment inhibitors of SOCS3 (expression or activity) allow sustainedp-STAT3 levels, and SOCS3 inhibition may be measured by STAT3activation. For example, COS cells can be treated with CNTF andmonitored for sustained phosph-STAT3 signals. In another embodiment,cultured neurons can be incubated in serum-free medium with or withoutserially-diluted inhibitor, e.g. for 6 hr. The cells are then incubatedwith a polyclonal antibody against phospho-STAT3, such as Tyr705 (CellSignaling Technology, Danvers, Mass.); see, e.g. Liu et al., J Neurosci,September 2001, 21(17) RC164, 1-5.

The use of various combinations of SOCS3 inhibitors is also envisioned.

Administration

Administration is to a subject by a route that results in contacting aneffective amount of the respective inhibitors to the target neuron(s).As the term is used herein, the target neuron is the neuron which isintentionally contacted by the administered agent. A target neuron canbe a lesioned neuron or a non-lesioned neuron (e.g., for compensatoryaxonal outgrowth to a region of dennervation). The target neuron may becontacted at one or more specific target sites of the neuron. As theterm is used herein, the target site of the neuron is the region of theneuron to which the agent is intentionally contacted. Regions of theneuron include the dendrites, cell body, and the axon. Sinceregeneration and axonal generation in the treatment of a neuronal injuryincludes compensatory promotion of axonal outgrowth of uninjuredneurons, benefit is expected from mere delivery of the inhibitors and/orother agents to an injury site. As such, suitable target neurons areactual damaged neurons, and also neurons that are in the immediate areaof an injury site or an area of dennervation. The specific location andextent of an injury site can be determined by the skilled practioner.Examples of injury sites are the site of physical damage or disruptionof neuronal activity. The immediate area of an injury site will varywith respect to the specific injury, the nature of the injury, and thenature of the injured neurons (e.g., axonal length, specific function,etc.) and can be determined by the skilled practitioner. Typically alesion is in the axon of the injured neuron. In one embodiment, theimmediate area of the injury site is within about 1-10 mm of identifieddamaged neurons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm).

In one embodiment, the administration is localized so as to be highlytargeted to a specific site. In one embodiment, the administration issystemic, and results in delivery of the appropriate concentration tothe specific site.

Depending on the intended route of delivery, the compositions may beadministered in one or more dosage form(s) (e.g. liquid, ointment,solution, suspension, emulsion, tablet, capsule, caplet, lozenge,powder, granules, cachets, douche, suppository, cream, mist, eye drops,gel, inhalant, patch, implant, injectable, infusion, etc.). The dosageforms may include a variety of other ingredients, including binders,solvents, bulking agents, plasticizers etc.

In a specific embodiment, the inhibitors are contacted with the neuronusing an implantable device that contains the inhibitors and that isspecifically adapted for delivery to a neuron. Examples of devicesinclude solid or semi-solid devices such as controlled releasebiodegradable matrices, fibers, pumps, stents, adsorbable gelatin (e.g.Gelfoam), etc. The device may be loaded with premeasured, discrete andcontained amounts of the inhibitors sufficient to promote sustainedregeneration or sustained survival of the neuron. In one embodiment, thedevice provides continuous contact of the neuron with the inhibitors atnanomolar or micromolar concentrations, (e.g., for at least 2, 5, or 10days, or for at least 2, 3, or 4 weeks, or for greater than 4 weeks,e.g., 5, 6, 7, or 8 weeks).

In one embodiment, administration of the inhibitor of PTEN and inhibitorof SOCS3 to a subject (e.g., in a single or in different pharmaceuticalcompositions, with or without an additional factor described herein)results in the inhibitors directly contacting an injured neuron in needof regeneration. In one embodiment, administration results in contactingneurons proximal to a site of neuronal injury. Neurons can be contactedat any point along their length (e.g., at the axon, dendrite and/or thecell body).

Administration to the subject can be by any one or combination of avariety of methods (e.g., parenterally, enterally and/or topically). Theappropriate method(s) will depend upon the circumstances of theindividual (e.g. the location of the target neuron(s), the condition ofthe individual, the desired duration of the contact, whether local orsystemic treatment is desired). The administration can be by any methodsdescribed herein that will result in contact of sufficient inhibitor(s)to the target neuron to promote sustained survival, sustainedregeneration, or a combination of both. For instance, parenteral,enteral and topical administration can be used. The phrases “parenteraladministration” and “administered parenterally” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subarachnoid, intraspinal, intracerebro spinal, and intrasternalinjection and infusion. Enteral administration involves the esophagus,stomach, and small and large intestines (i.e., the gastrointestinaltract). The phrases “systemic administration,” “administeredsystemically”, “peripheral administration” and “administeredperipherally” as used herein mean the administration of a compound otherthan directly into the central nervous system, such that it enters theanimal's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration. Administration maybe topical (including ophthalmic, vaginal, rectal, intranasal,epidermal, and transdermal), oral or pulmonary administration, e.g., byinhalation or insufflation, or intracranial, e.g., intrathecal orintraventricular, administration, topically to the eye, or byintraocular injection.

Specific routes of administration and the dosage regimen will bedetermined by skilled clinicians based on factors such as the exactnature of the condition being treated, the severity of the condition,and the age and general physical condition of the patient.

The invention also provides methods for promoting sustained survival,sustained regeneration, or a combination of both in a lesioned neuron ofcentral nervous system neurons following an injury. The method involvesadministering to a subject a combination of the inhibitor of PTEN andinhibitor of SOCS3 to the subject to thereby contact the site of injury.

The term “administering” to a subject includes dispensing, delivering orapplying an active compound in a pharmaceutical formulation to a subjectby any suitable route for delivery of the active compound to the desiredlocation in the subject to thereby contact the desired portion(s) of theneuron(s), (e.g., the injury, the injured neuron, or the site of desiredoutgrowth of the neuron). This includes, without limitation, delivery byeither the parenteral or oral route, intramuscular injection,subcutaneous/intradermal injection, intravenous injection, buccaladministration, transdermal delivery and administration by the rectal,colonic, vaginal, intranasal or respiratory tract route, intraocular,ocular. Another form of administration suitable for treatment of spinalcord injury is injection into the spinal column or spinal canal.

In one embodiment, the inhibitor(s) is contacted in vivo by introductioninto the central nervous system of a subject, e.g., into thecerebrospinal fluid of the subject. In certain aspects of the invention,the inhibitor(s) is introduced intrathecally, e.g., into a cerebralventricle, the lumbar area, or the cisterna magna. In another aspect,the inhibitor(s) is introduced intraocullarly, to thereby contactretinal ganglion cells or the optic nerve. Modes of administration aredescribed in U.S. Pat. No. 7,238,529.

In one embodiment, administration occurs following neuronal injury inthe subject, not prior to or at the time of neuronal injury.

In another embodiment of the invention, the inhibitor(s) formulation isadministered into a subject intrathecally. As used herein, the term“intrathecal administration” is intended to include delivering aninhibitor(s) formulation directly into the cerebrospinal fluid of asubject, by techniques including lateral cerebroventricular injectionthrough a burrhole or cisternal or lumbar puncture or the like(described in Lazorthes et al. Advances in Drug Delivery Systems andApplications in Neurosurgery, 143-192 and Omaya et al., Cancer DrugDelivery, 1: 169-179, the contents of which are incorporated herein byreference). The term “lumbar region” is intended to include the areabetween the third and fourth lumbar (lower back) vertebrae. The term“cisterna magna” is intended to include the area where the skull endsand the spinal cord begins at the back of the head. The term “cerebralventricle” is intended to include the cavities in the brain that arecontinuous with the central canal of the spinal cord. Administration ofan inhibitor(s) to any of the above mentioned sites can be achieved bydirect injection of the inhibitor(s) formulation or by the use ofinfusion pumps. For injection, the inhibitor(s) formulation of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the inhibitor(s) formulation may be formulated insolid form and re-dissolved or suspended immediately prior to use.Lyophilized forms are also included. The injection can be, for example,in the form of a bolus injection or continuous infusion (e.g., usinginfusion pumps) of the inhibitor(s) formulation.

In one embodiment of the invention, said inhibitor(s) formulation isadministered by lateral cerebro ventricular injection into the brain ofa subject in the inclusive period from the time of the injury to a timedetermined by the skilled practitioner (e.g., 100 hours). The injectioncan be made, for example, through a burr hole made in the subject'sskull. In another embodiment, said encapsulated therapeutic agent isadministered through a surgically inserted shunt into the cerebralventricle of a subject in the inclusive period from the time of theinjury to a time determined by the skilled practitioner (e.g., 100 hoursthereafter). For example, the injection can be made into the lateralventricles, which are larger, even though injection into the third andfourth smaller ventricles can also be made.

In yet another embodiment, said inhibitor(s) formulation is administeredby injection into the cisterna magna, or lumbar area of a subject in theinclusive period from the time of the injury to a time determined by theskilled practitioner (e.g., 100 hours thereafter). Administration can becontinuous, or can be by repeated doses. In one embodiment, the repeateddoses are formulated so that an effective amount of the inhibitors iscontinually present at the injury site.

Duration and Levels of Administration

The pharmaceutical composition, used in the method of the invention,contains a therapeutically effective amount of the inhibitor of PTENand/or SOCS3. A “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired result (e.g., sustained neuronal survival, sustained neuronaloutgrowth from lesioned or proximal neurons). A therapeuticallyeffective amount of the inhibitor may vary according to factors such asthe disease state, age, and weight of the subject, and the ability ofthe inhibitor (alone or in combination with one or more other agents) toelicit a desired response in the subject. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the inhibitor(s) thereof are outweighed by the therapeuticallybeneficial effects.

The term “therapeutically effective amount” refers to an amount that issufficient to effect a therapeutically reduction in a symptom associatedwith the neuronal injury, disease, disorder or condition describedherein, when administered to a typical subject who has said injury,disease, disorder, condition. A therapeutically significant reduction ina symptom is, e.g. about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%,about 150% or more as compared to a control or non-treated subject. Insome embodiments the term “therapeutically effective amount” refers tothe amount that is safe and sufficient to prevent or delay thedevelopment and further spread of neuronal injury, disease, or otherdisease symptoms. The amount can also cure or cause the disease,disorder or condition to go into remission, slow the course of, orotherwise inhibit progression by promoting sustained survival, sustainedregeneration, or a combination of both of the lesioned or threatenedneurons.

In one embodiment, the therapeutically effective amount is evidenced bythe restoration of nerve function. Restoration of nerve function can beevidenced, for example, by restoration of nerve impulse conduction, adetectable increase in conduction action potentials, observation ofanatomical continuity, restoration of more than one spinal root level,an increase in behavior or sensitivity, or a combination thereof.

Contacting of the injured neuron(s) (e.g., by administration to asubject) can be anytime following the injury. In one embodiment, theinjured neuron is contacted within 96 hours of formation of the lesionon the neuron to be contacted, and more preferably within 72, 48, 24, or12 hours. In one embodiment, the subject is administered one or bothinhibitors prior to injury as a precautionary measure.

The treatment of a subject may likewise begin anytime following theinjury. In one embodiment, the treatment progresses upon detection orsuspicion of the injury. For example, the treatment can be begun atabout 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 11hr, 12, hr, 18 hr, or 24 hours post injury. Benefit is also expected tobe had from treatment that takes place considerably longer after theinjury. The injury may have occurred more than three months prior to thetreatment, more than one month prior, more than three weeks prior to thetreatment, or more than two weeks prior to the treatment, more than oneweek prior to the treatment or from between 1-6 days prior to thetreatment.

Since the combined action of the inhibitors produces sustained survivaland sustained regeneration in a lesioned neuron, significant benefit isexpected to result from extended contacting of the inhibitors to thelesioned neuron. Such contacting can be achieved by extendedadministration of the inhibitors to the subject in need. As such,administration can take place for at least 2, 5, or 10 days. Even longerperiod of time are expected to also provide substantial benefit (e.g.,for at least 2, 3, or 4 weeks). In some situations administration forgreater than 4 weeks (e.g., 5, 6, 7, or 8 weeks) is expected to providetherapeutic results.

In one embodiment, the inhibitors (e.g., in the form of a pharmaceuticalcomposition) described herein are contacted to the neuron, and/oradministered to the subject in the period from the time of injury (forexample within 24, 12, 6, 3, or 1 hours after the injury has occurred)to for at least 2, 5, or 10 days, or for at least 2, 3, or 4 weeks, orfor greater than 4 weeks, e.g., 5, 6, 7, or 8 weeks). Useful longerperiod can be determined by the skilled practitioner.

In one embodiment, administration occurs following neuronal injury inthe subject, not prior to or at the time of neuronal injury. In oneembodiment, administration occurs prior to injury, as a precautionarymeasure.

Detection of Therapeutic Effects

The methods described herein can further comprise the further step ofdetecting a resultant regeneration of the axon. For in vitroapplications, axonal regeneration may be detected by any routinely usedmethod to assay axon regeneration such as a neurite outgrowth assay.

In one embodiment, the method of treatment further comprises a detectingstep, such as the step of detecting a resultant improved recovery fromthe injury, or detecting a resultant promoted regeneration of theinjured neuron. Such improvement can be detected directly using imagingmethodologies such as MRI, or indirectly or inferentially, such as byneurological examination showing improvement in the targeted neuralfunction. The detecting step may occur at any time point afterinitiation of the treatment, e.g. at least one day, one week, one month,three months, six months, etc. after initiation of treatment. In certainembodiments, the detecting step will comprise an initial neurologicalexamination and a subsequent neurological examination conducted at leastone day, week, or month after the initial exam. Improved neurologicalfunction at the subsequent exam compared to the initial exam indicatesresultant axonal regeneration. The specific detection and/or examinationmethods used will usually be based on the prevailing standard of medicalcare for the particular type of neuron injury being evaluated (i.e.trauma, neurodegeneration, etc.).

Pharmaceutically Acceptable Compositions

In one embodiment, the combination of inhibitors of PTEN and SOCS3 whichis administered in vivo to a subject is contained in one or morepharmaceutically acceptable compositions. The pharmaceutical compositionor solution can further include one or more other exogenous agents(e.g., one or more axogenic factors) described herein as administeredwith or contacted in the presence of the inhibitors. The pharmaceuticalcomposition may optionally be specifically formulated to exclude one ormore such other agents. In one embodiment, the pharmaceuticalcomposition consists essentially of the inhibitors of PTEN and SOCS3 anda pharmaceutically acceptable carrier. By the term “consists orconsisting essentially of” is meant that the pharmaceutical compositiondoes not contain any other active agents (e.g., modulators of neuronaloutgrowth).

A pharmaceutical composition comprising an effective amount of aninhibitor of SOCS3 and an effective amount of an inhibitor of PTEN isencompassed by the present invention. The pharmaceutical compositioncomprises the respective inhibitors in the respective concentrationsthat are sufficient to promote sustained survival, sustainedregeneration, or a combination of both to a lesioned neuron whenadministered at the appropriate dosage for the appropriate period oftime, as discussed herein.

In one embodiment, the pharmaceutical composition of the invention canbe provided as a packaged formulation. The packaged formulation mayinclude a pharmaceutical composition of the invention in a container andprinted instructions for administration of the composition for treatinga subject having a neuronal injury, and/or disease, disorder orcondition associated with neuronal injury, as described herein.

Pharmaceutical compositions are considered pharmaceutically acceptablefor administration to a living organism. For example, they are sterile,the appropriate pH, and ionic strength, for administration. Theygenerally contain the inhibitor(s) formulated in a composition within/incombination with a pharmaceutically acceptable carrier, also known inthe art as excipients.

The pharmaceutically acceptable carrier is formulated such that itfacilitates delivery of the active ingredient (e.g., the PTEN and SOCS3inhibitors) to the target site. Such a carrier is suitable foradministration and delivery to the target neuron. The pharmaceuticallyacceptable carrier will depend upon the location of the target neuronand the route of administration. For example, a typical carrier forintravenous administration of an agent is saline. The term“pharmaceutically acceptable carrier” includes, without limitation, anyand all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. For example, the carrier can besuitable for injection into the cerebrospinal fluid. The pharmaceuticalcomposition can further be designed to provide protection of theinhibitor from unnecessary dispersion or degredation. The pharmaceuticalcomposition may also contain additional ingredients such as stabilizersand disintegrants. Appropriate carriers and pharmaceutical compositionswill be determined by the skilled practitioner.

In one embodiment, the pharmaceutical composition is easily suspended inaqueous vehicles and introduced through conventional hypodermic needlesor using infusion pumps. Prior to introduction, the composition can besterilized with, preferably, gamma radiation or electron beamsterilization, described in U.S. Pat. No. 436,742 the contents of whichare incorporated herein by reference.

Additional examples of carriers are synthetic or natural polymers in theform of macromolecular complexes, nanocapsules, microspheres, or beads,and lipid-based formulations including oil-in-water emulsions, micelles,mixed micelles, synthetic membrane vesicles, and resealed erythrocytes.

In one embodiment, the pharmaceutically acceptable carrier comprises apolymeric matrix. The terms “polymer” or “polymeric” are art-recognizedand include a structural framework comprised of repeating monomer unitswhich is capable of delivering the inhibitor(s) such that treatment of atargeted condition, e.g., a nervous system injury, occurs. The termsalso include co-polymers and homopolymers e.g., synthetic or naturallyoccurring. Linear polymers, branched polymers, and cross-linked polymersare also meant to be included.

For example, polymeric materials suitable for forming the pharmaceuticalcomposition employed in the present invention, include naturally derivedpolymers such as albumin, alginate, cellulose derivatives, collagen,fibrin, gelatin, and polysacchanides, as well as synthetic polymers suchas polyesters (PLA, PLGA), polyethylene glycol, poloxomers,polyanhydrides, and pluronics. These polymers are biocompatible with thenervous system, including the central nervous system, they arebiodegradable within the central nervous system without producing anytoxic byproducts of degradation, and they possess the ability to modifythe manner and duration of the inhibitor(s) release by manipulating thepolymer's kinetic characteristics. As used herein, the term“biodegradable” means that the polymer will degrade over time by theaction of enzymes, by hydrolytic action and/or by other similarmechanisms in the body of the subject. As used herein, the term“biocompatible” means that the polymer is compatible with a livingtissue or a living organism by not being toxic or injurious and by notcausing an immunological rejection.

Polymers can be prepared using methods known in the art (Sandler. S. R.;Karo, W. Polymer Syntheses; Harcourt Brace: Boston. 1994; Shalaby, W.;Ikada, Y.; Langer, R.: Williams, J. Polymers of Biological andBiomedical Significance (ACS Symposium Series 540; American ChemicalSociety: Washington, D.C. 1994). Polymers can be designed to beflexible; the distance between the bioactive side-chains and the lengthof a linker between the polymer backbone and the group can becontrolled. Other suitable polymers and methods for their preparationare described in U.S. Pat. Nos. 5,455,044 and 5,576,018, the contents ofwhich are incorporated herein by reference.

The polymeric formulations are preferably formed by dispersion of theinhibitor(s) within liquefied polymer, as described in U.S. Pat. No.4,883,666, the teachings of which are incorporated herein by referenceor by such methods as bulk polymerization, interfacial polymerization,solution polymerization and ring polymerization as described in OdianG., Principles of Polymerization and ring opening polymerization, 2nded., John Wiley & Sons, New York, 1981, the contents of which areincorporated herein by reference. The properties and characteristics ofthe formulations are controlled by varying such parameters as thereaction temperature, concentrations of polymer and inhibitor, types ofsolvent used, and reaction times.

One or both of the inhibitors can be encapsulated in one or morepharmaceutically acceptable polymers, to form a microcapsule,microsphere, or microparticle, terms used herein interchangeably.Microcapsules, microspheres, and microparticles are conventionallyfree-flowing powders consisting of spherical particles of 2 millimetersor less in diameter, usually 500 microns or less in diameter. Particlesless than 1 micron are conventionally referred to as nanocapsules,nanoparticles or nanospheres. For the most part, the difference betweena microcapsule and a nanocapsule, a microsphere and a nanosphere, ormicroparticle and nanoparticle is size; generally there is little, ifany, difference between the internal structure of the two. In one aspectof the present invention, the mean average diameter is less than about45 μm, preferably less than 20 μm, and more preferably between about 0.1and 10 μm.

In another embodiment, the pharmaceutical composition compriseslipid-based formulations. Any of the known lipid-based drug deliverysystems can be used in the practice of the invention. For instance,multivesicular liposomes (MVL), multilamellar liposomes (also known asmultilamellar vesicles or “MLV”). unilamellar liposomes, including smallunilamellar liposomes (also known as unilamellar vesicles or “SUV”) andlarge unilamellar liposomes (also known as large unilamellar vesicles or“LUV”), can all be used so long as a sustained release rate of theencapsulated inhibitor(s) can be established. In one embodiment, thelipid-based formulation can be a multivesicular liposome system. Methodsof making controlled release multivesicular liposome drug deliverysystems is described in PCT Application Ser. Nos. US96/11642, US94/12957and US94/04490, the contents of which are incorporated herein byreference. The composition of the synthetic membrane vesicle is usuallya combination of phospholipids, usually in combination with steroids,especially cholesterol. Other phospholipids or other lipids may also beused.

Examples of lipids useful in synthetic membrane vesicle productioninclude phosphatidylglycerols, phosphatidylcholines,phosphatidylserines, phosphatidylethanolamines, sphingolipids,cerebrosides, and gangliosides. Preferably phospholipids including eggphosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol areused.

In preparing lipid-based vesicles containing inhibitor(s), suchvariables as the efficiency of encapsulation, lability of the inhibitor,homogeneity and size of the resulting population of vesicles,inhibitor-to-lipid ratio, permeability, instability of the preparation,and pharmaceutical acceptability of the formulation should be considered(see Szoka, et al., Annual Reviews of Biophysics and Bioengineering,9:467, 1980; Deamer, et al., in Liposomes, Marcel Dekker, New York,1983, 27; and Hope, et al., Chem. Phys. Lipids, 40:89, 1986, thecontents of which are incorporated herein by reference). [0059] In one,the pharmaceutical composition provides sustained delivery, e.g., “slowrelease” of the inhibitor(s) to a subject for at least one, two, three,or four weeks after the pharmaceutical composition is administered tothe subject.

As used herein, the term “sustained delivery” is intended to includecontinual delivery of the inhibitors in vivo over a period of timefollowing administration, preferably at least several days, a week orseveral weeks. Sustained delivery of the inhibitors can be demonstratedby, for example, the continued therapeutic effect of the inhibitors overtime (e.g., by continued outgrowth of neurons over time). Alternatively,sustained delivery of the inhibitors may be demonstrated by detectingthe presence of the inhibitors in vivo over time.

In one embodiment, the pharmaceutical composition provides sustaineddelivery of the inhibitor(s) thereof to a subject for less than 30 daysafter the inhibitor(s) is administered to the subject. For example, thepharmaceutical composition, e.g., “slow release” formulation, canprovide sustained delivery of the inhibitor(s) to the subject for one,two, three or four weeks after the formulation is administered to thesubject. Alternatively, the pharmaceutically composition may providesustained delivery of the inhibitor(s) to a subject for more than 30days after the formulation is administered to the subject.

Other Agents

The PTEN inhibitor and SOCS3 inhibitor can be contacted to the injuredneuron in combination with, or prior or subsequent to, other agents(also referred to herein as additional agents) such as anti-inflammatoryor anti-scarring agents, growth or trophic factors, denervation-inducedcytokines, etc. In one embodiment, the lesion results from acute spinalcord injury and the method additionally comprises contacting the neuronwith methylprednisolone sufficient to reduce inflammation of the spinalcord. In one embodiment, the inhibitors are administered in combinationwith trophic and/or growth factors (e.g., denervation-induced cytokines)known in the art to promote or enhance neuornal survival/regeneration,growth and/or differentiation. Examples include, without limitation,brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor(CNTF) (WO2011/066182), fibroblast growth factor (FGF), chondroitiniase,nerve growth factor (NGF), NT-3 (Piantino et al, Exp Neurol. 2006October; 201(2):359-67), inosine (Chen et al, Proc Natl Acad Sci USA.(2002) 99:9031-6; U.S. Pat. No. 6,551,612 to Benowitz; U.S. Pat. No.6,440,455 to Benowitz; and US Pat Publ 20050277614 to Benowitz),oncomodulin (Yin et al, Nat Neurosci. (2006) 9:843-52; US Pat Publ20050054558 to Benowitz; US Pat Publ 20050059594 to Benowitz; and U.S.Pat. No. 6,855,690 to Benowitz). Another such agent is an agent toremove extracellular matrix molecules (e.g., chondroitin sulphateproteoglycans) that are inhibitory to neuronal outgrowth, such aschondroitenase ABC (ChABC), which breaks up chondroitin sulphateproteoglycans.

In one embodiment, the inhibitors are administered in combination withone or more factors that facilitate neuronal synapse formation. Examplesof such factors include, without limitation, activators of Rab3A,NMDA-I, synapsin-1, tetanus toxin receptor, BDNF-receptor and a GABAreceptor. Such factors are described in U.S. Patent ApplicationPublication 2008/0214458. Neuronal synapse formation can be modulated,for example, by modulating the activity of the transcriptional factormyocyte enhancer factor 2 (MEF2) (e.g., MEF2A), MEF2C, MEF2D, dMEF2,CeMEF2, Activating transcription factor 6 beta (ATF6), Estrogen relatedreceptor alpha (ERR1), Estrogen related receptor beta (ERR2), Estrogenrelated receptor gamma (ERR3), Erythroblastosis virus E26 oncogenehomolog 1 (ETS1), Forkhead box protein C2 (FOXC2), Gata binding factor 1(GATA-1), Heat shock factor 1 (HSF1), HSF4, MLL3, Myeloblastosisoncogene homolog (MYB), Nuclear receptor coactivator 2 (NCOA2), Nuclearreceptor corepressor 1 (NCOR1), Peroxisome proliferative activatedreceptor gamma (PPARg), SMAD nuclear interacting protein 1 (SNIP1),SRY-box containing protein 3 (SOX3), SOX8, SOX9, Sterol regulatoryelement-binding transcription factor 2 (SREBP2), or Thyroid hormonereceptor beta-1 (THRB1) (described in U.S. Patent ApplicationPublication 20100112600).

The other agent(s) can be administered to the same site or to adifferent site as the PTEN inhibitor and/or SOCS3 inhibitor. The otheragent may be contacted to the same site of the neuron or to a differentsite of the neuron. In one embodiment, the PTEN inhibitor and/or theSOCS3 inhibitor is contacted to the neuron(s) at the neuron's region oforigin in the brain (e.g., by administration to cortical neurons at thecerebral ventricle) and the other agent is contacted to the neuron atthe site of injury (e.g., the lesioned axon such as a cortical spinaltract axon). Other combinations of site of contact and routes ofadministration discussed herein are also envisioned.

Devices

The invention also provides activator-eluting or activator-impregnatedimplantable solid or semi-solid devices. Examples of implantable devicesinclude polymeric microspheres (e.g. see Benny et al., Clin Cancer Res.(2005) 11:768-76) or wafers (e.g. see Tan et al., J Pharm Sci. (2003)4:773-89), biosynthetic implants used in tissue regeneration afterspinal cord injury (reviewed by Novikova et al., Curr Opin Neurol.(2003) 6:711-5), biodegradable matrices (see e.g. Dumens et al.,Neuroscience (2004) 125:591-604), biodegradable fibers (see e.g. U.S.Pat. No. 6,596,296), osmotic pumps, stents, adsorbable gelatins (seee.g. Doudet et al., Exp Neurol. (2004) 189:361-8), etc. Preferreddevices are particularly tailored, adapted, designed or designated forimplantation. The implantable device may contain one or more additionalagents used to promote or facilitate neural regeneration. For example,in one embodiment, an implantable device used for treatment of acutespinal cord injury contains the activator and methylprednisolone orother anti-inflammatory agents. In another embodiment, the implantabledevice contains the activator and a nerve growth factor, trophic factor,or hormone that promotes neural cell survival, growth, and/ordifferentiation, such as brain-derived neurotrophic factor (BDNF),ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), inosine,oncomodulin, NT-3, etc.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means±1%.

In one respect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising). In some embodiments, other elements tobe included in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

The present invention may be as defined in any one of the followingnumbered paragraphs.

-   1. A method of promoting sustained survival in a lesioned mature    neuron, sustained regeneration in a lesioned mature neuron,    sustained compensatory outgrowth in a mature neuron, or a    combination thereof, comprising:    contacting the neuron with an effective amount of an inhibitor of    PTEN and an effective amount of an inhibitor of SOCS3 to thereby    promote sustained survival, sustained regeneration, and/or sustained    compensatory outgrowth of the neuron.-   2. The method of paragraph 1, wherein the lesioned mature neuron is    the result of an acute injury.-   3. The method of paragraph 2, wherein the acute injury is selected    from the group consisting of crush, severing, and acute ischemia.-   4. The method of paragraph 1, wherein the lesioned mature neuron is    the result of chronic neurodegeneration.-   5. The method of any one of paragraphs 1-4 wherein contacting first    occurs within 24 hours of the injury-   6. The method of any one of paragraphs 1-4, wherein contacting first    occurs within 3 days of the injury.-   7. The method of any one of paragraphs 1-4, wherein contacting first    occurs within 6 days of the injury.-   8. The method of any one of paragraphs 1-7, wherein contacting is    continued for a period of time selected from the group consisting of    1 week after initiation, 2 weeks after initiation 3 weeks after    initiation, 4 weeks after initiation, 5 weeks after initiation, 6    weeks after initiation, 7 weeks after initiation, and 8 weeks after    initiation.-   9. The method of any one of paragraphs 1-8, wherein contacting    occurs in vivo.-   10. The method of any one of paragraphs 1-8, wherein contacting    occurs in vitro.-   11. The method of any one of paragraphs 1-10, wherein the neuron is    human.-   12. A method of treating a subject for a CNS lesion, comprising:    administering to the subject a therapeutically effective amount of    an inhibitor of PTEN and a therapeutically effective amount of an    inhibitor of SOCS3, wherein administering results in contacting one    or more target CNS neurons of the subject with the inhibitor of PTEN    and the inhibitor of SOCS3, to thereby promote sustained survival,    sustained regeneration, sustained compensatory outgrowth, or a    combination thereof in the CNS neurons.-   13. The method of paragraph 12, wherein the subject is a human.-   14. The method of any one of paragraphs 1-13, wherein the inhibitor    of PTEN is selected from the group consisting of:-   (a) potassium bisperoxo(bipyridine)oxovanadate (V) (bpV(bipy));-   (b) dipotassium    bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (V) (bpV(HOpic));-   (c) potassium bisperoxo(1,10-phenanthroline)oxovanadate (V),    (bpV(phen));-   (d) dipotassium bisperoxo(picolinato)oxovanadate (V), (bpV(pic));    and-   (e) combinations thereof.-   15. The method of any one of paragraphs 1-14, wherein the inhibitor    of SOCS3 is selected from the group consisting of SOCS3-specific    hpRNA, siRNA, antisense SOCS3, dominant negative SOCS3, and    combinations thereof.-   16. The method of any one of paragraphs 12-15, wherein the CNS    lesion results from an acute injury.-   17. The method of paragraph 16, wherein the acute injury is selected    from the group consisting of crush, severing, and acute ischemia.-   18. The method of any one of paragraphs 16-17 wherein administration    first occurs within 24 hours of the injury-   19. The method of any one of paragraphs 16-17, wherein    administration first occurs within 3 days of the injury.-   20. The method of any one of paragraphs 16-17, wherein    administration first occurs within 6 days of the injury.-   21. The method of paragraph 12, wherein the CNS lesion results from    chronic neurodegeneration.-   22. The method of paragraph 12, wherein the CNS lesion results from    a traumatic injury.-   23. The method of paragraph 12 wherein the CNS lesion results from a    traumatic brain injury.-   24. The method of paragraph 12 wherein the CNS lesion results from a    stroke.-   25. The method of paragraph 12 wherein the lesioned CNS neuron is in    the optic nerve.-   26. The method of paragraph 12 wherein the CNS lesion results from    an acute spinal cord injury.-   27. The method of paragraph 12 wherein the lesioned CNS neuron is in    the spinal cord of a patient, and the inhibitor is intrathecally    administered to the patient.-   28. The method of paragraph 12 wherein lesioned CNS neuron is a    sensory neuron.-   29. The method of any one of paragraphs 1-28 wherein the inhibitor    is administered intravenously.-   30. The method of any one of paragraphs 1-28 wherein the inhibitor    is administered intrathecally.-   31. The method of any one of paragraphs 1-28 wherein the inhibitor    is administered ocularly.-   32. The method of any one of paragraphs 1-28 wherein the inhibitor    is administered locally at the neuron.-   33. The method of any one of paragraphs 1-32, wherein an additional    agent is administered to the subject.-   34. The method of paragraph 33, wherein the additional agent is    selected from the group consisting of inosine, oncomodulin, BNDF,    NGF, CNTF, and combinations thereof.-   35. A device for promoting sustained survival of a lesioned mature    neuron, sustained regeneration of a lesioned mature neuron,    compensatory outgrowth of a neuron, or a combination thereof,    comprising a reservoir loaded with a premeasured and contained    amount of a therapeutically effective amount of an inhibitor of PTEN    and an inhibitor of SOCS3, and specifically adapted for implementing    the method of paragraph 12.-   36. A pharmaceutical composition comprising a therapeutically    effective amount of an inhibitor of SOCS3 and a therapeutically    effective amount of an inhibitor of PTEN.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1

During development axons reach their targets first through de novooutgrowth in embryos, followed by “networked growth” in which axonselongate with termini tethered to their targets. As animals increase inbody size during postnatal and adolescent stages, the distance resultedfrom the “networked growth” could be much longer than that traveled bythe initial de novo growth. After injury in the adult CNS, regeneratingaxons need to carry out de novo growth over relatively vast distances toreach their targets. Thus, the robustness of axon regeneration, in termsof both speed and duration of axon regrowth, is critical for makingfunctional reconnections in adulthood. Approaches that have been shownto promote axon regeneration in the adult CNS include reducingextracellular inhibitory activity and increasing intrinsic growthability¹⁻⁹. However, the extents of axon regeneration observed in thesestudies are still limited. For example, previous studies demonstratedthat the injured optic nerve could undergo significant axon regenerationafter conditional deletion of PTEN or SOCS3 in adult RGCs, but theregrowth only occurred during the first 2 weeks post-injury, and thensubsided afterwards^(1,2).

Results

To identify a strategy for promoting sustained robust axon regeneration,the effects of deleting both PTEN and SOCS3 in adult RGCs on optic nerveregeneration was assessed. Adeno-associated viruses (AAV)-Cre (AAV-GFPas a control) were injected into the vitreous body of PTEN^(f/f(10)), orSOCS3^(f/f(11)), or PTEN^(f/f)/SOCS3^(f/f) mice to delete the floxedgenes 2 weeks prior to optic nerve injury. In addition, ciliaryneurotrophic factor (CNTF) was applied intravitreously to theSOCS3^(f/f) or PTEN^(f/f)/SOCS3^(f/f) mice, as this enhances axonregeneration induced by SOCS3 deletion².

At 2 weeks post-injury, a significant increase in axon regeneration inthe double knockout group was observed (FIG. 6 a-b). The synergisticeffects of the double deletion became even more dramatic at 4 weeksafter injury (FIG. 1). At 2 mm distal to the lesion site, deletion ofboth genes resulted in more than 10-fold increase in the number ofregenerating axons compared to deletion of either gene alone (FIG. 1a-d). In the double mutants, more than 20% of the regenerating axonsreached the region proximal to the optic chiasm (FIG. 1c ). Among theregenerating axons passing the chiasm, some crossed the midline andprojected to the contralateral side, while others remained ipsilateral.Occasionally, a few axons could be seen projecting into the oppositeuninjured optic nerve (FIG. 1c ). Interestingly, several regeneratingaxons could grow even further, reaching the optic tract brain entry zoneand in the hypothalamus, the suprachiasmatic nuclei area (FIG. 7).

Compared to wild type animals, all three mutant groups showedsignificantly increased RGC survival after injury. At 2 weeks afterinjury, the survival was comparable among the three mutant groups (FIG.6c ). At 4 weeks after injury however, the number of surviving RGCsdeclined in the PTEN or SOCS3 single mutants, while the survival ratewas maintained in the double mutants (FIG. 1e ).

To mimic a clinically relevant scenario, whether a delayed deletion ofPTEN and/or SOCS3 still promoted sustained optic nerve regeneration wasexamined. Thus, intravitreal AAV-Cre injection was performed immediatelyafter optic nerve injury (FIG. 2). It takes at least 3-6 days forCre-dependent reporter expression to be observed (FIG. 8a ). RGCsurvival and axon regeneration was examined 3 weeks post-injury in theseanimals. Despite similar survivals in all groups (FIG. 8b-c ), doubleand single mutants showed significant differences in the extents of axonregeneration (FIG. 2). At 0.5 mm distal to the lesion site, up to20-fold more regenerating axons were seen in double mutants, compared toindividual single mutants. Thus, adult RGCs with concomitant deletion ofPTEN and SOCS3 can activate a program for sustained de novo axon growthafter injury.

What mechanism(s) contribute to the synergy produced by concurrentdeletion of PTEN and SOCS3 was then examined. While mTOR activation islikely to be a major mediator of PTEN deletion¹, the regenerationphenotype of SOCS3 deletion is dependent on gp130, a shared receptorcomponents for cytokines^(12,13). However, multiple down-streameffectors have been implicated in cytokines-gp130 signaling¹²⁻¹⁵.Because of a suggested relevance to axon regeneration¹⁶⁻¹⁸, the specificinvolvement of the transcription factor STAT3, a major target of theJAK/STAT pathway¹⁹ was tested. Upon phosphorylation-mediated activation,STAT3 accumulates in the nucleus to initiate transcription¹⁹. Byimmunostaining with anti-phospho-STAT3, phospho-STAT3 expression wasfound to be rarely detectable in intact RGCs. In wild type mice, opticnerve injury increased phospho-STAT3 levels in RGCs, but such signalswere mainly localized in the cytosol (FIG. 3a, b ). In contrast,phospho-STAT3 was evident in the nuclei of axotomized RGCs with bothSOCS3 single and SOCS3/PTEN double mutants (FIG. 3a, b ), suggesting theactivation of STAT3 under these conditions.

The contribution of STAT3 to the axon regeneration induced by SOCS3deletion and CNTF administration was then evaluated. Deletion of STAT3had no significant effects on RGC survival (FIG. 9) and axonregeneration (FIG. 3c ). However, double deletion of STAT3 and SOCS3abolished injury-induced phospho-STAT3 signal (FIG. 3a, b ), RGCsurvival (FIG. 9), and axon regeneration (FIG. 3c, d ) seen in SOCS3single mutants, suggesting that STAT3 is a critical mediator of SOCS3regulated axon regeneration and RGC survival.

Possible interactions of the PTEN- and SOCS3-regulated pathways on opticnerve regeneration were then examined. Similar to wild type,phospho-STAT3 expression in PTEN deleted RGCs after injury was rarelydetectable (FIG. 10), arguing against STAT3 activation after PTENdeletion. Importantly, the extents of axon regeneration and RGC survivalwere similar in the animals with PTEN single deletion and PTEN/STAT3 orPTEN/gp130 double deletion (FIG. 4a, c and FIG. 11a, b ), suggestingthat STAT3 is unlikely to be an important mediator of PTEN deletion.

The potential role of mTOR activation in axon regeneration induced bySOCS3 deletion was also evaluated. While systematic administration ofrapamycin, a specific mTOR inhibitor, abolished the majority of the axonregeneration after PTEN deletion (FIG. 4a, c ), the same treatment didnot affect axon regeneration from SOCS3-deleted RGCs (FIG. 4b, d ).These results suggest that these two pathways act independently inregulating axon regeneration.

To assess possible gene expression alteration triggered by PTEN/SOCS3double-deletion, gene-expression profiling studies were performed.Transgenic YFP17 mice expressing YFP in most RGCs (with only fewamacrine cells, FIG. 12a ), either in a control background or crossed tothree different mutants, were subjected to AAV-Cre injection and opticnerve injury. 3-days post injury, mRNAs were extracted from FACS-sortedRGCs and analyzed by microarray (FIG. 12b-e ).

The first potential mechanism is that certain key regeneration-promotinggenes are significantly altered by PTEN/SOCS3 double deletion, whencompared to both single deletions and the wild type controls. Among 15genes selected, two encode critical positive mTOR regulators, namelysmall GTPase Rheb and Insulin-like growth factor 1 (IGF1)²⁰ (FIG. 13,16), suggesting that positive feedback regulation of the mTOR activityin the double mutant may contribute to the enhanced and sustained axonregeneration. The list also includes several axon growth-related genes,such as the RNA-binding protein Elav14 (HuD), the cell adhesionmolecules MAM domain-containing glycosylphosphatidylinositol anchor 2(Mdga2)²¹, procadherin beta 9 (Pcdhb9)²², the axon guidance moleculeUnc5D²³, and a cAMP-regulator phosphodiesterase 7B (Pde7b)²⁴ (FIG. 13,16).

In addition, double deletion may “enhance” the regeneration-relatedgene-expression changes that occur poorly or moderately in the singlemutants. By the criteria of significant changes (q>0.05, foldchange<1.6) for comparisons between the double mutants and wild typecontrols, but not between the single mutants and wild type controls, thegene set shown in FIG. 14a was revealed. This includes most of genesshown in FIG. 13. Further, it shows the up-regulation of a number ofaxon growth-related genes, such as the signaling moleculemitogen-activated protein kinase kinase (Map2k4)²⁵ and the axontransport components dynein component Dync1li2, kinesin family memberKif21a, and kinesin-associated protein 3 (Kifap3)^(26,27) (FIG. 14a ,16). Consistently, pathway analysis indicates that this list is enrichedin genes serving cellular functions related to axon growth (FIG. 14b ).

Other non-exclusive possibilities may also contribute to the synergy ofthe double deletion. For example, SOCS3 deletion might regulate certainaxon growth-promoting genes that are poorly regulated by PTEN singledeletion, thus, double deletion allows the actions of both mTOR activityand these genes. Therefore screening was performed for two sets of genespreferentially regulated by SOCS3 or PTEN deletion in the doubledeletion induced gene alteration (FIG. 15). These lists contain a numberof genes related to axon regeneration, but whether any of the abovegenes show complementary/synergistic functions is still unknown. Inaddition, Kruppel-like factors KLF4 and KLF6 showed expression changesin opposite directions (although the changes of KLF4 did not reachstatistical significance, FIG. 16), consistent with proposed oppositefunctions of these regeneration regulators⁸.

Complementarily, the expression of a subset of genes in both intact andinjured RGCs was assessed by in situ hybridization. When compared to theexpression in intact RGCs, some genes, such as Elav14 and KLF6, wereinduced in the mutant(s) after injury (FIG. 17a, b ), consistent withthe model of the activation of axon growth-related genes in thesemutants. However, some other genes, such as axon transport genesDync1li2, Kif21a, and Kifap3, and a transcription factor ZFP40, weremaintained in the double-mutant RGCs but down-regulated in both wildtype and single mutant RGCs after injury (FIG. 17c-f ). These resultssuggested that in addition to inducing growth-related gene expression,the double deletion enables injured neurons to maintain their pre-injuryphysiological states, which might be an important contributing mechanismfor the enhanced and sustained axon regeneration.

Together, these experiments reveal an important strategy for achievingsustainable de novo axon regrowth in the adult CNS neurons:co-activation of specific protein translations and gene transcriptionsby concomitant inactivation of PTEN and SOCS3. Notably, the mTORactivity is maintained and phospho-STAT3 levels are increased in adultperipheral sensory neurons after injury^(17,28). Thus, the activationstates of these two pathways may underlie the differential regenerativeabilities of CNS and PNS neurons. However, deletion of PTEN and SOCS3 isnot converting the CNS neurons to a PNS-like state, because PTEN issimilarly expressed in adult PNS neurons and SOCS3 is increased duringPNS regeneration^(17,29). Nonetheless, enhancing mTOR activity throughdeletion of PTEN or TSC2 also drastically increases axon re-growth inPNS neurons^(29,30), indicating deletion of PTEN and SOCS3 may make anend-run around different growth-suppressive mechanisms. Considering theformidable long distances that regenerating axons must travel in theadult after injury, the synergistic effects of two different pathwayssuggest a potential solution to this challenge, making the goal offunctional recovery more realistic.

Materials and Methods

Summary of Methods

AAV-Cre Injection and Optic Nerve Injury. Adult mice were intravitreallyinjected with AAV-Cre and/or CNTF to the left eyes. Optic nerve injuryand quantifications were done with the methods describedpreviously^(1,2).

Purification of RGCs. 72 hours after injury, isolated retinas wereincubated in digestion solution, dissociated by gentle trituration, andthen filtered before FACS sorting.

RNA Extraction and Microarray. Isolated RNA was subjected to microarrayanalysis. Data were log 2 transformed at probe level, and the PMmodel-based expression values were annotated and normalized using dChip.Statistical significance of gene expression differences between groupswas determined by SAM (Significance Analysis of Microarrays). After aninitial filter of very low expressed genes (average log 2 transformedvalue <5), a False Discovery Rate (FDR rate), or q value, less than 0.05were used to generate the significant gene lists. Functional analyseswere performed using DAVID (The Database for Annotation, Visualizationand Integrated Discovery).

Detailed Methods

AAV-Cre Injection. All experimental procedures were performed incompliance with animal protocols approved by the IACUC at Children'sHospital, Boston. C57BL6/J mice (WT) or various foxed mice includingRosa-lox-STOP-lox-Tomato (from F. Wang), SOCS3^(f/f), PTEN^(f/f),SOCS3^(f/f)/PTEN^(f/f), STAT3^(f/f), gp130^(f/f),SOCS3^(f/f)/STAT3^(f/f), PTEN^(f/f)/STAT3^(f/f), PTEN^(f/f)/gp130^(f/f),YFP-17 crossed with or without SOCS3^(f/f) and/or PTEN^(f/f) wereintravitreally injected with 1-2 μl volume of AAV-Cre (titers at0.5-1.0×10¹²) to the left eyes. For each intravitreal injection, a glassmicropipette was inserted into the peripheral retina, just behind theora serrata, and was deliberately angled to avoid damage to the lens. Insome experiments, 1 μl (1 μg/μl) CNTF (Pepro Tech) was intravitreallyinjected immediately after injury and at 3 days post injury, and weeklythereafter.

Optic Nerve Injury. Two weeks following AAV-Cre injection, the leftoptic nerve was exposed intraorbitally and crushed with jeweler'sforceps (Dumont #5; Roboz) for 5 seconds approximately 1 mm behind theoptic disc. Counting TUJ1+ RGCs in retina whole mount and regeneratingaxons were done with the methods described previously^(1,2).

Purification of RGCs. YFP17 mice, either themselves or crossed with PTENand/or SOCS3 floxed mice, were subjected to AAV-Cre injection and opticnerve injury. 72 hours post injury, animals were euthanized and retinaswere immediately dissected out for dissociation. Retinas were incubatedin digestion solution (20 U/ml papain, Worthington; 1 mM L-cysteine HCL;0.004% DNase; 0.5 mM EDTA in Neurobasal) for 30 min at 37° C., and thenmoved into Ovomucoid/BSA (1 mg/ml) solution to stop digestion.Subsequently, retinas were dissociated by gentle trituration withtrituration buffer (0.5% B27; 0.004% DNase; 0.5 mM EDTA in Opti-MEM),and then filtered through 40 μm cell strainer (BD Falcon) before FACSsorting.

FACS sorting was performed with BD FACSAria Hu. Each time immediatelybefore sorting, a purity test was performed to insure the specificityfor sorting YFP signal is higher than 99%. Dissociated retinal cellswere separated based on both size (forward scatter) and surfacecharacteristics (side scatter). Aggregated cells were excluded based onFSC-H vs FSC-A ratio. Retinal cells without YFP expression was used asnegative controls to set up the detection gate each time before sortingthe YFP positive cells. Sorted cells were immediately performed with RNAextraction.

RNA Extraction and Microarray. RNA was extracted using the Qiagen RNeasymini kit (Qiagen), and RNA quality was assessed using a bioanalyzer(Agilent Technologies). For microarray assays, RNA was amplified andlabeled with the Nugen Ovation WTA System (Nugen), to obtain 2.8 μg ofcRNA to be hybridized on Affymetrix mouse Genechip 1.0 ST (Affymetrix).To ensure reproducibility and biological significance, threehybridizations were performed for each group, with RNA samples collectedfrom three independent FACS purifications, each including three or fouranimals (biological replicates). The microarray data accession number isGSE32309

Data from microarrays were log 2 transformed at probe level, and the PMmodel-based expression values were annotated and normalized using dChip(www.dchip.org <http://www.dchip.org/>). Statistical significance ofgene expression differences between groups was determined by SAM(Significance Analysis of Microarrays) software(http://www-stat.stanford.edu/˜tibs/SAM/). After an initial filter ofvery low expressed genes (average log 2 transformed value <5), a FalseDiscovery Rate (FDR rate), or q value, less than 0.05 were used togenerate the significant gene lists.

Functional annotation clustering analysis was performed using DAVID (TheDatabase for Annotation, Visualization and Integrated Discovery,http://david.abcc.ncifuf gov/). The functional annotation groups withsimilar EASE score, the Fish Exact Probability Value, were clustered andgrouped under the same overall enrichment score. The higher of thescore, the more enriched. The five top-scored clusters were listed, andthe count of genes and their percentage to corresponding categories inthe database were shown.

Example 2

Central nervous system lesions, such as spinal cord injury and stroke,can damage projecting neurons, resulting in the de-nervation offunctional important target areas. In principle, if the neuronal cellbody is spared, functional recovery can be achieved by regeneration oflesioned axons; alternatively, especially when neuronal cell bodies arelost, sprouting from non-injured neurons/axons can form new circuitscompensating for the lost functions. Although spontaneous sprouting canoccur extensively in early postnatal life, it is restricted in theadult, thus functional deficits are often permanent.

The corticospinal pathway, controlling voluntary movements, isparticularly important for functional recovery after spinal cord injuryand stroke. It is also a valuable model for studying injury-induced axonsprouting in rodents, because it has a precise topographic organizationof fibers projecting into the spinal cord, which is lost withoutcompensation after a simple pyramidotomy in wild type animals. Thismouse corticospinal injury model was therefore utilized to examinewhether SOCS3 or SOCS3 PTEN double deletion could promote compensatorysprouting from the uninjured contralateral side.

Results

The results of the experiment are presented in FIG. 5. In resultsobtained from wild type mice (Left panel) BDA labeled CST fibers wererarely seen in the denervated side of spinal cord. In results obtainedfrom SOCS3^(−/−) mice (central panel), the SOCS3 deletion promotedrobust CST sprouting into the denervated side of the spinal cord. Inresults obtained from SOCS3^(−/−) and PTEN^(−/−) mice (right panel), theSOCS3 and PTEN double deletion synergistically promoted CST sprouting.Sprouting was observed at the C6-8 spinal cord level.

These results indicate co-inhibition of PTEN and SOCS3 synergize atpromoting compensatory sprouting from intact and spared axons afterpartial injury, which is different from promoting axon regeneration frominjured neurons. These data provide a basis for designing therapies forincomplete injuries such as stroke, traumatic brain injury, multiplesclerosis and spinal cord injury.

Materials and Methods

AAV Injection. Neonatal SOCS3^(loxP/loxP) orSOCS3^(loxP/loxP)/PTEN^(loxP/loxP) mice were cryoanesthetized andinjected with 2 μl of AAV-Cre for gene deletion or AAV-GFP as control.Injections were made into the right sensorimotor cortex using ananoliter injector attached to a fine glass pipette. Mice were thenplaced on a warming pad and returned to their mothers after regainingnormal color and activity. A pyramidotomy was performed 6-8 weeks later.

Pyramidotomy. Mice were anesthetized with ketamine/xylazine. An incisionwas made at the left side of the trachea. Blunt dissection was performedto expose the skull base and a craniotomy in the occipital bone allowedfor access to the medullary pyramids. The left or right pyramid was cutwith a fine scalpel medially up to the basilar artery. The wound wasclosed in layers with 6.0 sutures. The mice were placed on soft beddingon a warming blanket held at 37° C. until fully awake.

BDA Tracing. 8-12 weeks after injury, CST axons on the non-injured sidewas anterogradely traced with biotinylated dextran amines (BDA). A totalof 4.0 μl of BDA (10%, Invitrogen) was injected into sensorimotor cortexat four sites (anterior-posterior coordinates from bregma in mm:1.0/1.5, 0.5/1.5, −0.5/1.5, −1.0/1.5, all at a depth of 0.5 mm intocortex). Mice were kept for an additional 2 weeks before being killed.

REFERENCES FOR EXAMPLES 1 AND 2

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Neuroprotective and axon growth-promoting    effects following inflammatory stimulation on mature retinal    ganglion cells in mice depend on ciliary neurotrophic factor and    leukemia inhibitory factor. J Neurosci 29, 14334-14341 (2009).-   8. Moore, D. L., et al. KLF family members regulate intrinsic axon    regeneration ability. Science 326, 298-301 (2009).-   9. Winzeler, A. M., et al. The lipid sulfatide is a novel    myelin-associated inhibitor of CNS axon outgrowth. J Neurosci 31,    6481-6492 (2011).-   10. Groszer, M., et al. Negative regulation of neural    stem/progenitor cell proliferation by the Pten tumor suppressor gene    in vivo. Science 294, 2186-2189 (2001).-   11. Mori, H., et al. Socs3 deficiency in the brain elevates leptin    sensitivity and confers resistance to diet-induced obesity. Nat Med    10, 739-743 (2004).-   12. Fasnacht, N. & Muller, W. Conditional gp130 deficient mouse    mutants. Semin Cell Dev Biol 19, 379-384 (2008).-   13. Ernst, M. & Jenkins, B. J. Acquiring signalling specificity from    the cytokine receptor gp130. Trends Genet 20, 23-32 (2004).-   14. Park, K. K., et al. Cytokine-induced SOCS expression is    inhibited by cAMP analogue: impact on regeneration in injured    retina. Mol Cell Neurosci 41, 313-324 (2009).-   15. Park, K., Luo, J. M., Hisheh, S., Harvey, A. R. & Cui, Q.    Cellular mechanisms associated with spontaneous and ciliary    neurotrophic factor-cAMP-induced survival and axonal regeneration of    adult retinal ganglion cells. J Neurosci 24, 10806-10815 (2004).-   16. Bareyre, F. M., et al. In vivo imaging reveals a phase-specific    role of STAT3 during central and peripheral nervous system axon    regeneration. Proc Natl Acad Sci USA 108, 6282-6287 (2011).-   17. Miao, T., et al. Suppressor of cytokine signaling-3 suppresses    the ability of activated signal transducer and activator of    transcription-3 to stimulate neurite growth in rat primary sensory    neurons. J Neurosci 26, 9512-9519 (2006).-   18. Qiu, J., Cafferty, W. B., McMahon, S. B. & Thompson, S. W.    Conditioning injury-induced spinal axon regeneration requires signal    transducer and activator of transcription 3 activation. J Neurosci    25, 1645-1653 (2005).-   19. Aaronson, D. S. & Horvath, C. M. A road map for those who don't    know JAK-STAT. Science 296, 1653-1655 (2002).-   20. Sengupta, S., Peterson, T. R. & Sabatini, D. M. Regulation of    the mTOR complex 1 pathway by nutrients, growth factors, and stress.    Mol Cell 40, 310-322 (2010).-   21. Joset, P., et al. Rostral growth of commissural axons requires    the cell adhesion molecule MDGA2. Neural Dev 6, 22 (2011).-   22. Junghans, D., Haas, I. G. & Kemler, R. Mammalian cadherins and    protocadherins: about cell death, synapses and processing. Curr Opin    Cell Biol 17, 446-452 (2005).-   23. Low, K., Culbertson, M., Bradke, F., Tessier-Lavigne, M. &    Tuszynski, M. H. Netrin-1 is a novel myelin-associated inhibitor to    axon growth. J Neurosci 28, 1099-1108 (2008).-   24. Hannila, S. S. & Filbin, M. T. The role of cyclic AMP signaling    in promoting axonal regeneration after spinal cord injury. Exp    Neurol 209, 321-332 (2008).-   25. Nix, P., Hisamoto, N., Matsumoto, K. & Bastiani, M. Axon    regeneration requires coordinate activation of p38 and JNK MAPK    pathways. Proc Natl Acad Sci USA 108, 10738-10743 (2011).-   26. Hanz, S. & Fainzilber, M. Retrograde signaling in injured    nerve—the axon reaction revisited. J Neurochem 99, 13-19 (2006).-   27. Hoffman, P. N. A conditioning lesion induces changes in gene    expression and axonal transport that enhance regeneration by    increasing the intrinsic growth state of axons. Exp Neurol 223,    11-18 (2010).-   28. Park, K. K., Liu, K., Hu, Y., Kanter, J. L. & He, Z. PTEN/mTOR    and axon regeneration. Exp Neurol 223, 45-50 (2010).-   29. Abe, N., Borson, S. H., Gambello, M. J., Wang, F. & Cavalli, V.    Mammalian target of rapamycin (mTOR) activation increases axonal    growth capacity of injured peripheral nerves. J Biol Chem 285,    28034-28043 (2010).-   30. Christie, K. J., Webber, C. A., Martinez, J. A., Singh, B. &    Zochodne, D. W. PTEN inhibition to facilitate intrinsic regenerative    outgrowth of adult peripheral axons. J Neurosci 30, 9306-9315    (2010).

Example 3

Injury to the mammalian adult CNS often results in functional deficits,largely owing to the disruption of neuronal circuits. In the case ofspinal cord injury, the disruption of axonal tracts that conveyascending sensory and descending motor information leads to pronouncedand persistent sensorimotor dysfunction in the body below the lesion.Presumably, re-building the neuronal circuits may result from two typesof axon regrowth: regenerative growth of injured axons and/orcompensatory sprouting from spared axons. While spontaneous regenerativegrowth occurs rarely in the adult CNS, compensatory sprouting of thesame or different types of axons occurs often after incomplete injuryand has been proposed as a major mechanism for spontaneous functionalrecovery after CNS injuries (Kaas 1991; Florence et al., 1998; Maier andSchwab, 2008; Benowitz and Carmichael, 2010; Rosenzweig et al., 2010).

In both experimental animal models and human patients, partial injury ofthe spinal cord is often followed by functional recovery, which isusually incomplete (Maier and Schwab, 2008). For example, in monkeys, asmall portion (25%) of spared white matter is sufficient to allowrecovery of coordinated hindlimb locomotion but not grasping afterinjury (Eidelberg et al. 1981). Importantly, a close correlation hasbeen observed between the anatomical reorganization of spared descendingfibers and spontaneous functional recovery after injury. For example, inyoung animals with a unilateral CST lesion, CST fibers from theuninjured side sprout heavily into the contralateral, denervated side,and this sprouting is followed by a high level of recovery of forelimbfunction (Kuang & Kalil 1990; Rouiller et al. 1991; Aisaka et al. 1999).However, in adults, spared descending fibers send few to no collateralsacross the midline to the denervated side (Aoki et al. 1986; Woolf etal. 1992; Goldstein et al. 1997; Weidner et al., 2001; Bareyre et al.,2004; Cafferty and Strittmatter, 2006), and this has been proposed as akey limiting factor for functional recovery after spinal cord injury inthe adult.

In recent studies investigating the molecular mechanisms that controlthe intrinsic regenerative ability of mature CNS neurons, it has beenshown that the mTOR activity is a critical determinant of intrinsicgrowth ability and undergo a down-regulation in cortical neurons overthe course of postnatal development (Liu et al., 2010). As a result,deletion of PTEN, a negative regulator of the mTOR pathway (reviewed inMa and Blenis, 2009; Liu et al., 2011), significantly increasescompensatory sprouting responses of CNS neurons (Liu et al., 2010),suggesting that neuronal intrinsic growth ability is an importantregulator of axonal sprouting.

PTEN deletion in cortical neurons does not induce sprouting, however,unless there is also an injury (Liu et al., 2010), suggesting thatincreased intrinsic growth potential, by itself, is insufficient toinitiate a sprouting response. This observation raises an importantquestion: what extrinsic factors trigger spared axons to initiate asprouting response after partial injury? In this study, it wasdemonstrate that genetic deletion of SOCS3, an established inhibitor ofthe JAK/STAT pathway, enhances CST sprouting after unilateralpyramidotomy. The JAK/STAT pathway is usually activated by cytokinessuch as CNTF (Nicholson et al., 2000; Crocker et al., 2008).Consistently, it is also shown that denervated neurons in the spinalcord up-regulate CNTF expression, suggesting that denervation-inducedcytokine expression might be an important trigger for axonal sprouting.

Results

SOCS3 Deletion Increases CST Sprouting after Unilateral Pyramidotomy

Previous studies showed that deleting SOCS3 in retinal ganglion neuronspromotes the regeneration of injured optic nerve axons after injury(Smith et al., 2009, Sun et al., 2011). Thus, whether SOCS3 deletioncould affect the sprouting response of CST axons after unilateralpyramidotomy was assessed (FIG. 22G). In this injury paradigm, CST axonsfrom the left cortical hemisphere are transected at the medullarypyramid above the pyramidal decussation. To monitor collateral sproutingfrom uninjured CST axons, BDA is injected into the right sensoromotorcortex at 4 weeks post-injury and cross sections from different levelsof the spinal cord are analyzed after waiting an additional 2 weeks(FIG. 22G). In control mice, most of the labeled axons are on the leftside of the spinal cord, with minimal labeling on the right side(Weidner et al., 2001; Bareyre et al., 2004; Cafferty and Strittmatter,2006; Liu et al., 2010). Thus, the number of labeled axons on the rightside of the spinal cord originating from the left intact CST can be usedto quantify the extent of CST compensatory sprouting.

To delete SOCS3 in cortical neurons, Cre-expressing adeno-associatedvirus (AAV-Cre) was injected into the right side of the sensorimotorcortex of homozygous conditional SOCS3 mutants (SOCS3loxp/loxp, Mori etal., 2004) on postnatal day 1 (P1). This approach has been previouslyshown to induce efficient Cre-dependent recombination in neuronsthroughout the sensorimotor cortex (Liu et al., 2010). Deletion of SOC3at this stage did not appear to change the pattern of CST projections inthe adult (FIG. 18A). A left unilateral pyramidotomy was performed ateight weeks of age, and sprouting responses were analyzed 6 weekspost-injury. SOCS3 deletion significantly increased sprouting from thespared (intact, left) half of the CST into the denervated (right) sideof the spinal cord (FIG. 18C). The number of labeled CST axons isenhanced most significantly at cervical levels of the spinal cord, butthe increase is also obvious at lower levels (FIG. 22A-22F). In thedenervated (right) side of the spinal cord, the labeled axons could beseen in different regions of the gray matter, with the most abundantprojections in the intermediate and dorsal spinal cord (FIG. 18C). Thedensity of compensatory/collateral sprouting fibers amounted to 25% ofthe uncrossing CST (FIG. 18D), similar to the extent observed after PTENdeletion (Liu et al., 2010). These results suggest that the signalingpathway(s) regulated by SOCS3 regulate the capacity for compensatorysprouting of spared CST axons.

To determine if SOC3 deletion is able to enhance post-injury sproutingwhen it is deleted from neurons at stages later than P1, the aboveexperiment was repeated with a CamKII-Cre driver, which is not active inCST neurons until P21 (FIG. 23, Yu et al., 2001). As shown in FIG. 19,significant sprouting responses are also observed in SOCS3f/f micecrossed with CamKII-Cre mice, and the extent was similar to that seenwhen SOCS3 is deleted at P1 (compare FIGS. 18C and 19B). This suggeststhat SOCS3 deletion in neonatal and relatively mature cortical neuronsis able to initiate sprouting responses after a unilateral pyramidotomy.

CNTF is up-regulated in the spinal cord after unilateral pyramidotomySOCS3 is a negative regulator of the JAK/STAT pathway, which is oftenactivated by cytokines such as CNTF (Crocker et al., 2008; Sun et al.,2011). Enhanced CST sprouting from intact cortical neurons after SOCS3deletion suggests that axonal sprouting responses might be regulated byaccess to extrinsic cytokines which activate the JAK/STAT pathway.Because the pyramidotomy is performed on one side of the medullarypyramid, we examined the expression of CNTF in the cortex (where CSTaxons originate) and in the spinal cord (where CST axons terminate) inboth intact and injured wild type mice.

As shown in FIGS. 24G and 24H, no detectable immunoreactivity withanti-CNTF antibodies could be found on either side of the cortex after aleft pyramidotomy on different days post-injury, suggesting that it isunlikely that CNTF from cortical regions contribute to the enhancedsprouting response. However, at 3 days post-injury, CNTFimmunoreactivity was significantly increased in the spinal cord,especially on the denervated side, compared to the low level signalsseen in the intact spinal cord (FIG. 20).

CNTF is Up-regulated in Neurons after Pyramidotomy

Next, possible mechanisms of CNTF up-regulation after unilateral CSTablation were examined. At least two possibilities could be envisioned.First, neurons in the spinal cord deprived of CST inputs may up-regulatecytokines to stimulate collateral axonal sprouting. Second, aninflammatory response triggered by the Wallerian degeneration of thetransected CST might result in an upreguation of cytokines frominfiltrating immune cells as well as activated CNS cells, such asastrocytes and microglia. Indeed, although the lesion site for thepyramidotomy is at the medullary pyramid level and there are nomanipulations applied to the spinal cord, we found that by 3 days afterunilateral pyramidotomy, CD68+ cells (likely macrophages or microglia)accumulated in the spinal cord, with more in the right dorsal columnwhere the transected CST undergo Wallerian degeneration (FIG. 24C).Sparse GFAP labeling was seen in the intact spinal cord, and this is notaltered by a left pyramidotomy, suggesting no obvious astrocyteactivation after unilateral pyramidotomy (FIG. 24D).

Next, which cell type(s) increased CNTF expression in the spinal cordafter a left pyramidotomy was examined. It appeared that mostimmuno-reactivity with anti-CNTF antibodies co-localizes with NeuN+neurons (FIG. 20), but not with CD68+ or GFAP+ cells (FIGS. 24E and24F). This is different from what was seen after spinal cord injury,where CNTF expression is increased in reactive glial cells around thelesion (Tripathi and Mctigue, 2008). Thus, by using the pyramidotomy atthe medullary pyramid, our results reveal that CNTF expression isinduced mainly in neurons in the spinal cord after CST inputs aredepleted, which might provide a possible explanation for extensive CSTsprouting towards the denervated site. The identity of these neurons,however, is unknown. It is also unknown whether these neurons are director indirect synaptic targets of CST axons.

To assess whether the CNTF up-regulation in denervated neurons issecondary to the inflammatory response in the degenerating CST, the timecourse of the accumulation of CD68+ cells versus CNTF+up-regulation inneurons was analyzed. Increased CNTF signals were seen as early as 2 dayafter injury, which continues to increase at 3 days after injury (FIG.20). CD68+ cells, on the other hand, did not become obvious until 3 dayspost-injury (FIG. 24C and data not shown), arguing against the notionthat CNTF up-regulation in neurons is secondary to the inflammatoryresponse.

Exogenously Applied CNTF Triggers CST Sprouting in the Absence ofPyramidotomy

Next, whether exogenously applied CNTF is sufficient to trigger CSTsprouting in uninjured adult mice was examined. AAV-Cre was injectedinto SOCS3f/f mice at P1, and CNTF was injected into the spinal cord atC1 when the animals reached adulthood. CST sprouting was then analyzed 8weeks after injection. As shown in FIG. 25A-F, CNTF, but not saline,injected into the dorsal spinal cord at the C1 level, indeed triggered asignificant CST sprouting response at the C1 (FIG. 25A-D), but not C7(FIGS. 25E and 25F), spinal cord level. In these animals, many CSTsprouts crossed the midline and projected towards the contralateral sideof the spinal cord. However, the extent of sprouting is less than afterleft pyramidotomy. This may be due to the fact that the elevation ofCNTF after pyramidotomy might be more prolonged than what was achievedwith our single injection. Additionally, other cytokines or growthfactors might be involved in sprouting after unilateral pyramidotomy.

Further enhanced CST sprouting induced by co-deletion of SOCS3 and PTENThe results above suggest that denervation-triggered CNTF expression inneurons might be an important extrinsic regulator of CST collateralsprouting. Whether increasing intrinsic growth capacity in uninjured CSTneurons by increasing mTOR activity by a deletion of PTEN could furtherincrease the extent of CST sprouting elicited by SOCS3 deletion wasexamined. Either AAV-Cre or AAV-GFP was injected to the rightsensorimotor cortex of PTENf/f/SOCS3f/f mice at P1, performed thepyramidotomy on the left side at 8 weeks of age, and analyzed the extentof CST sprouting at 6 weeks post-injury.

In intact PTEN and SOCS3 double-deleted mice without pyramidotomy, theCST projection pattern remains unaltered (FIG. 21A). However, afterunilateral pyramidotomy, uninjured CST axons underwent a significantlyincreased sprouting response (FIG. 21C-21E, FIG. 26), compared with thatseen in the single SOCS3-deleted (FIG. 18C) or single PTEN-deleted (Liuet al., 2010) mice. As shown in FIG. 21D, the number of sprouting axonsobserved in the double mutants is more than what was seen in the singlemutants combined (see FIG. 18 and Liu et al., 2010) and is similar towhat was seen after unilateral pyramidotomy performed at P7 (Liu et al.,2010). In these double mutants, sprouting axons innervated almost all ofthe gray matter on the right side of the spinal cord, although theiroverall distribution is similar to that seen in the single mutants.These results suggest that the increased intrinsic growth abilityresulting from co-deletion of PTEN and SOCS3 greatly enhancescorticospinal neuron sprouting after a unilateral pyramidotomy.

Discussion

This study demonstrates that the sprouting response of the CST isregulated by a SOCS3-dependent signaling pathway in that SOCS3 deletionin cortical neurons enhances the sprouting response after unilateralpyramidotomy. This appears to be initiated, at least in part, by CNTFexpressed by those neurons on the side of the spinal cord that aredeprived of CST inputs. Furthermore, co-deletion of SOCS3 and PTENfurther enhances the sprouting of spared CST axons, allowing CST sproutsto occupy the entire empty field left by the injured half. Overall, thisstudy reveals a powerful new strategy for promoting functionalreinnervation after injury.

Sprouting-triggering Cytokines

Mechanistically, an important question regards the nature of thesignal(s) that leads to collateral growth from spared CST axons afterinjury. Previous studies suggest that the expression of many genes ischanged in the denervated spinal cord after unilateral pyramidotomy(Bareyre et al., 2002) and in cortical neurons that undergo axonalsprouting (Li et al., 2010), but it is unclear whether these are theprimary triggers for axonal sprouting or secondary to changes that occurduring or after axonal reorganization. The results strongly suggestedthat CNTF and perhaps other cytokines could be an important class oftriggers for CST collateral sprouting. Three lines of evidence supportthis notion: SOCS3 deletion in cortical neurons promotes CST sproutingafter injury; this injury-induced sprouting is correlated with CNTFexpression; and exogenously applied CNTF promotes sprouting inSOCS3-deleted neurons in the absence of an injury.

CNTF and other cytokines have been implicated in promoting neuronalsurvival and axonal growth in both PNS and CNS. In both dorsal rootganglion neurons (Bareyre et al., 2011) and retinal ganglion neurons(Smith et al., 2009, Sun et al., 2011) CNTF triggers axon regeneration,which is largely mediated by the transcription factor STAT3. The datapresented herein indicate that similar mechanisms also mediate the CSTsprouting after unilateral pyramidotomy. Interestingly, the sproutingtriggered by CNTF injection is less pronounced than what is seen afterinjury, suggesting possible involvements of other cytokines.

Extrinsic and Intrinsic Control of CST Sprouting

The herein presented dramatically enhanced CST sprouting response fromcorticospinal neurons with a co-deletion of SOCS3 and PTEN indicates afunctional interaction between these two signaling pathways. Because ofthe established role of SOCS3 as a negative regulator ofcytokine-activated JAK/STAT pathway, the effects of SOCS3 deletion arelikely triggered by extracelluar cytokines such as CNTF. In this aspect,a recent study showed that STAT3 selectively regulates initiation butnot later perpetuation of axonal growth in sensory neurons (Bareyre etal., 2011). On the other hand, PTEN deletion could act by enhancingneuronal mTOR activity, a likely determinant of neuronal intrinsicgrowth ability (Park et al., 2008, Liu et al., 2010). Without beingbound by theory, it is thought that SOCS3 deletion primes the neuron foran enhanced response to CNTF, and PTEN deletion acts synergistically tofurther enhance the intrinsic growth response to injury-induced signals.In axotomized retinal ganglion neurons, co-deletion of SOCS3 and PTENinduces the expression of IGF-1 and Rheb, two positive regulators of themTOR pathway, suggesting that a positive feedback mechanism might act tosustain mTOR activity in injured neurons (Sun et al., 2011). It would beinteresting for the future studies to find out whether similar ordifferent mechanisms are involved in the sprouting response of sparedaxons after unilateral pyramidotomy.

Denervated Neurons as a Source of Generating CNTF

Surprisingly, despite the inflammatory response associated withWallerian degeneration of transected CST axons, inflammatory cells donot show CNTF up-regulation. Instead, it was found that after unilateralpyramidotomy, neurons in the denervated spinal cord generate such asprout-promoting cytokine. Interestingly, CNTF-expressing neurons areconcentrated, but not limited to the termination territory of CST axons,suggesting that these CNTF-expressing neurons might be either direct orindirect targets of CST axons in intact animals. It is conceivable thatupon denervation these neurons might undergo electrophysiological and/orbiochemical alterations that lead to the induction of cytokineexpression. Further investigation of denervation-triggered cytokineup-regulation might reveal new insights into the mechanisms ofactivity-dependent structural reorganization.

In addition to spontaneous recovery from partial injury, axonalsprouting-mediated functional recovery also occurs after certainmanipulations such as rehabilitation. For example, robotic-basedtraining was shown to promote extensive reorganization of corticalprojections at the brainstem and spinal cord levels, allowing paralyzedrats to regain voluntary locomotion (van den Brand et al., 2012). Itwill be important to assess the possible involvement of activity-inducedcytokines in this and other types of functional recovery. These resultsprovide new insights into designing combinatorial strategies forpromoting functional recovery after injury.

Materials and Methods

Mice and Surgeries. All experimental procedures were performed incompliance with animal protocols approved by the Institutional AnimalCare and Use Committee at Children's Hospital, Boston. AAV, serotype 2,preparation was described previously (ref). For AAV injection, neonatalPten^(f/f), SOCS3^(f/f) or PTEN^(f/f)/SOCS3^(f/f) mice were anesthetizedand injected with 2 μl of either 10¹² GC/ml AAV-Cre or AAV-GFP into foursites of the right sensorimotor cortex using a nanoliter injectorattached to a fine glass pipette. Mice were then placed on a warming padand returned to their mothers after regaining normal color and activity.In other sets of experiments, CamkII-Cre/SOCS3^(f/f) mice were used.

pyramidotomy, mice were anesthetized with ketamine/xylazine. Theprocedure is similar to that described previously (ref). Briefly, anincision was made at the left side of the trachea. Blunt dissection wasperformed to expose the skull base and a craniotomy in the occipitalbone allowed for access to the medullary pyramids. The left pyramid wascut with a fine scalpel medially up to the basilar artery. The wound wasdosed in layers with 6.0 sutures. The mice were placed on soft beddingon a warming blanket held at 37° C. until fully awake. We traced theintact CST 4 weeks later with BDA (see below). For CNTF injection intoadult SOCS3 f/f mice with neonatal AAV-Cre cortical injection, alaminectomy was performed at C1 and 1 ul of CNTF (10 ug/ml, PeproTech,450-13) was injected with a nanoliter injector into the ventral side ofthe dorsal column (0.5 mm deep). BDA tracing was performed 6 weekslater.

BDA Tracing. To label CST axons by anterograde tracing, a total of 2.0μl of BDA (10%, invitrogen, D-1956) was injected into the rightsensorimotor cortex at five sites (anterior-posterior coordinates frombregma in mm: 1.0, 0.5, 0, −0.5, −1.0, all at 1.0 mm lateral and at adepth of 0.5 mm). Mice were kept for an additional 2 weeks before beingsacrificed.

Histology and Immunohistochemistry. Mice were given a lethal dose ofanesthesia and transcardially perfused with 4% paraformaldehyde. Brainsand spinal cords were isolated and post-fixed in the same fixativeovernight at 4° C. Tissues were cryoprotected via increasingconcentrations of sucrose. After embedding into OCT compound, thesamples were snap frozen in dry ice. Serial sections (25 μm) werecollected and stored at −20° C. until processed. Coronal sections of thelower medulla were cut for counting BDA-labeled CST fibers. Forassessing the extent of CST sprouting, serial sections at C7 and otherlevels of the spinal cords were cut in the transverse plane.

Immunostaining was performed following standard protocols. Allantibodies were diluted in a solution consisting of 5% normal goat ordonkey serum (NGS) and 1% Triton X-100 in phosphate-buffered saline(PBS). We used goat antibodies to CNTF (5 μg/ml, R&D Systems), ratantibodies to CD68 (1:200, Serotec), rabbit antibody to GFAP (1:200,Wako) and mouse antibody to NeuN (1:100, Millipore). Sections wereincubated with primary antibodies overnight at 4° C. and washed threetimes for 10 min with PBS. Secondary antibodies (Biotin-conjugateddonkey antibody to goat and Alexa 488-conjugated goat antibody torabbit, rat and mouse) were then applied and incubated for 1 h at 20-25°C. For CNTF staining, Elite Avidin biotin Conjugate (ABC, Vector Lab)was applied, followed by ISA Cyanine 3 (perkin Elmer). To detectBDA-labeled fibers, BDA staining was performed by incubating thesections in PBS containing streptavidin-horseradish peroxidase. Theremaining staining procedure was performed according to the protocolprovided by ISA Cyanine 3 system (Perkin Elmer).

Axonal Counting and Quantifications. For the groups of pyramidotomy,digital images of C7 or other levels of the spinal cord transversesections were collected using a Nikon fluorescence microscope.Densitometry measurement on each side of the gray matter was taken usingMetamorph software, after being subthresholded to the background andnormalized by area. The outcome measure of the sprouting density indexwas the ratio of contralateral and ipsilateral counts.

To quantify the number of sprouting axons, the methods used in previousstudies (Liu et al., 2010) were followed. Briefly, a horizontal line wasdrawn through the central canal and across the lateral rim of the graymatter. Three vertical lines were then drawn to divide the horizontalline into three equal parts, starting from the central canal to thelateral rim. Only fibers crossing the three lines were counted in eachsection. The results were presented after normalization with the numberof counted CST fibers at the medulla level. At least three sections werecounted for each mouse.

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What is claimed:
 1. A pharmaceutical composition for promoting sustainedsurvival in an injured neuron, sustained regeneration in an injuredneuron, and sustained compensatory outgrowth in an injured neuron,comprising a therapeutically effective amount of an inhibitor ofsuppressor of cytokine signaling (SOCS3) and a therapeutically effectiveamount of an inhibitor of phosphatase and tensin homolog (PTEN).
 2. Thepharmaceutical composition of claim 1, wherein the inhibitor of PTEN isselected from the group consisting of: (a) potassiumbisperoxo(bipyridine)oxovanadate (V) (bpV(bipy)); (b) dipotassiumbisperoxo(5-hydroxypyridine-2-carboxyl)oxoyanadate (V) (bpV(HOpic)); (c)potassium bisperoxo(1,10-phenanthroline)oxovanadate (V), (bpV(phen));(d) dipotassium bisperoxo(picolinato)oxovanadate (V), (bpV(pic)); and(e) combinations thereof.
 3. The pharmaceutical composition of claim 1,wherein the inhibitor of SOCS3 is selected from the group consisting ofSOCS3-specific hpRNA, siRNA, antisense SOCS3, dominant negative SOCS3,and combinations thereof.
 4. The pharmaceutical composition of claim 1,further comprising an agent selected from the group consisting ofinosine, oncomodulin, BNDF, NGF, CNTF, and combinations thereof.
 5. Thepharmaceutical composition of claim 1 that is formulated for ocularadministration.
 6. The pharmaceutical composition of claim 1 that isformulated for intravenous administration.
 7. The pharmaceuticalcomposition of claim 1 that is formulated for intrathecaladministration.
 8. A device for promoting sustained survival in aninjured neuron, sustained regeneration in an injured neuron, andsustained compensatory outgrowth in an injured neuron comprising areservoir loaded with a premeasured and contained amount of apharmaceutical composition comprising a therapeutically effective amountof an inhibitor of phosphatase and tensin homolog (PTEN) and atherapeutically effective amount of an inhibitor of suppressor ofcytokine signaling (SOCS3).
 9. The device of claim 8, wherein theinhibitor of PTEN is selected from the group consisting of: (a)potassium bisperoxo(bipyridine)oxovanadate (V) (bpV(bipy)); (b)dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (V)(bpV(HOpic)); (c) potassium bisperoxo(1,10-phenanthroline)oxovanadate(V), (bpV(phen)); (d) dipotassium bisperoxo(picolinato)oxovanadate (V),(bpV(pic)); and (e) combinations thereof.
 10. The device of claim 8,wherein the inhibitor of SOCS3is selected from the group consisting ofSOCS3-specific hpRNA, siRNA, antisense SOCS3, dominant negative SOCS3,and combinations thereof.
 11. The device of claim 8, wherein thepharmaceutical composition further comprises an agent selected from thegroup consisting of inosine, oncomodulin, BNDF, NGF, CNTF, andcombinations thereof.
 12. The device of claim 8, wherein thepharmaceutical composition is formulated for ocular administration. 13.The device of claim 8, wherein the pharmaceutical composition isformulated for intravenous administration.
 14. The device of claim 8,wherein the pharmaceutical composition is formulated for intrathecaladministration.
 15. The device of claim 8, which is implantable.
 16. Thepharmaceutical composition of claim 1, wherein the inhibitors aresurrounded by a polymeric matrix designed to provide protection of therespective inhibitor from dispersion and/or degradation.
 17. Thepharmaceutical composition of claim 1, wherein the inhibitors areformulated for sustained delivery.
 18. The pharmaceutical composition ofclaim 1, that is in a container.
 19. The pharmaceutical composition ofclaim 1, that is contained in an administration device for injection.